Heterologous targeting peptide grafted aavs

ABSTRACT

The disclosure in some aspects relates to recombinant adeno-associated viruses having distinct tissue targeting capabilities. In some aspects, the disclosure relates to gene transfer methods using the recombinant adeno-associated viruses. In some aspects, the disclosure relates to isolated AAV capsid proteins and isolated nucleic acids encoding the same.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional patent application U.S. Ser. No. 62/059,738, filed on Oct.3, 2014, and entitled, “HETEROLOGOUS TARGETING PEPTIDE GRAFTED AAVS”,the entire content of which is incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under RO1 NS066310awarded by the NIH/NINDS. The government has certain rights in theinvention.

FIELD

In some aspects, the disclosure provides novel AAVs and methods of usethereof as well as related kits.

BACKGROUND

Adeno-associated virus (AAV) is a small (˜26 nm) replication-defective,nonenveloped virus, that depends on the presence of a second virus, suchas adenovirus or herpes virus, for its growth in cells. AAV wasdiscovered in 1960s as a contaminant in adenovirus (a cold causingvirus) preparations. Its growth in cells is dependent on the presence ofadenovirus and, therefore, it was named as adeno-associated virus. AAVcan infect both dividing and non-dividing cells and may incorporate itsgenome into that of the host cell. These features make AAV a veryattractive candidate for creating viral vectors for gene therapy.

SUMMARY

Aspects of the disclosure relate to recombinant AAVs (rAAVs) engineeredto contain heterologous targeting peptides that target the rAAVs tocertain cells and/or tissues. RAAVs disclosed herein are useful becausethey can effectively deliver nucleic acids of interest to a particularcell and/or tissue, e.g., for purposes of manipulating levels of aparticular gene product in the cell and/or tissue. For example, in someembodiments, the disclosure provides rAAVs comprising a capsid proteinhaving a heterologous targeting peptide that confers unique tissuetargeting and cell transduction properties. In some embodiments,heterologous targeting peptides are provided that are represented by theformula [A]_(n), wherein A is alanine and n is an integer in a range of5 to 31. In some embodiments, such heterologous targeting peptides areuseful for targeting AAVs to tissues of the central nervous system(CNS). In some embodiments, heterologous targeting peptides are providedthat are in a range of 11 to 31 amino acids in length. In someembodiments, heterologous targeting peptides are provided that are in arange of 11 to 27 amino acids in length. In some embodiments,heterologous targeting peptides are provided that are in a range of 17to 21 amino acids in length. In some embodiments, heterologous targetingpeptides are provided that are 19 amino acids in length. In someembodiments, a heterologous targeting peptide is a polypeptiderepresented by SEQ ID NO: 5. In some embodiments, a heterologoustargeting peptide is represented by SEQ ID NO: 7. In some embodiments, aheterologous targeting peptide is a polypeptide encoded by the nucleicacid sequence represented by SEQ ID NO: 29.

In some embodiments, a heterologous targeting peptide further comprisesan N-terminal tag. In some embodiments, the N-terminal tag is a peptidetag. In some embodiments, the N-terminal tag is a Myc tag. In someembodiments, the N-terminal tag is a poly-histidine tag (His) tag. Insome embodiments, the tag comprises consecutive histidines in the rangeabout 2 amino acids to about 10 amino acids, about 6 amino acids toabout 10 amino acids, about 6 amino acids to about 20 amino acids, orabout 6 amino acids to about 30 amino acids in length. In someembodiments, the His tag comprises or consists of six amino acids inlength. In some embodiments, the His tag is has six amino acids and isreferred to as a hex-His tag.

In some embodiments, a capsid protein having a heterologous targetingpeptide is a VP1 capsid protein. In some embodiments, a capsid proteinhaving a heterologous targeting peptide is a VP2 capsid protein. In someembodiments, a capsid protein having a heterologous targeting peptide isa VP3 capsid protein. In some embodiments, an rAAV comprises a VP1and/or VP2 and/or VP3 capsid protein comprising a heterologous targetingpeptide. In some embodiment, the heterologous targeting peptide isN-terminally grafted in or to the capsid protein. In some embodiments, aAAV capsid protein further comprises a linker conjugated to theC-terminus of the heterologous targeting peptide. In some embodiments,the linker comprises a stretch of two or more glycine residues. In someembodiments, the polypeptide repeat is GGGGS (SEQ ID NO: 28).

In some embodiments, the heterologous targeting peptide targets the CNS,optionally the brain. In some aspects, the disclosure relates to an rAAVcomprising an AAV capsid protein having a heterologous targeting peptide(e.g., which is N-terminally grafted in or to the capsid protein) thatmediates transcytosis across the blood-brain barrier. In someembodiments, the heterologous targeting peptide is a lipoproteinreceptor-related protein (LRP) ligand.

In some embodiments, the disclosure relates to an AAV capsid proteinhaving a heterologous targeting peptide, in which the AAV capsid proteinis not of an AAV2 serotype. In some embodiments, an AAV capsid proteinhaving a heterologous targeting peptide is a VP2 capsid protein. In someembodiments, an AAV capsid protein having a heterologous targetingpeptide is of a serotype derived from a non-human primate. In someembodiments, the AAV capsid protein having a heterologous targetingpeptide is of a AAVrh8 serotype. In some embodiments, an AAV capsidprotein having a heterologous targeting peptide is of an AAV9,optionally AAV9.47, serotype.

In some embodiments, an N-terminally grafted heterologous targetingpeptide is inserted before the first, second, third, fourth, fifth,sixth, seventh, eighth, ninth, or tenth amino acid of the capsidprotein. In some embodiments, an N-terminally grafted heterologoustargeting peptide is inserted between the first and second N-terminalamino acids of the capsid protein. In some embodiments, the firstN-terminal amino acid of the capsid protein (e.g., a VP1, VP2 or V3capsid protein) is a methionine. Accordingly, in some embodiments, anN-terminally grafted heterologous targeting peptide may be insertedafter (e.g., immediately after) an N-terminal methionine residue of acapsid protein. In some embodiments, the first N-terminal amino acid ofthe capsid protein (e.g., a VP2 capsid protein) is an amino acid encodedby a non-canonical start codon, such as a threonine. Accordingly, insome embodiments, an N-terminally grafted heterologous targeting peptidemay be inserted after (e.g., immediately after) an N-terminal threonineresidue of a capsid protein.

In some embodiments, the disclosure relates to a rAAV comprising acapsid protein having an N-terminally grafted heterologous targetingpeptide, in which the N-terminally grafted heterologous targetingpeptide is present only in the VP2 capsid protein. In some embodiments,the disclosure relates to a composition comprising a rAAV comprising acapsid protein having an N-terminally grafted heterologous targetingpeptide, in which the N-terminally grafted heterologous targetingpeptide is present only in the VP2 capsid protein. In some embodiments,the composition further comprises a pharmaceutically acceptable carrier.In some embodiments, an rAAV comprises a capsid protein having an aminoacid sequence selected from the group consisting of: SEQ ID NOs: 19-27.

In some embodiments, the disclosure relates to a host cell containing anucleic acid that comprises a coding sequence selected from the groupconsisting of: SEQ ID NOs: 8-18 that is operably linked to a promoter.In some embodiments, the disclosure relates to a composition comprisingthe host cell described above. In some embodiments, the compositioncomprising the host cell above further comprises a cryopreservative.

The disclosure also relates to methods of delivering a transgene to asubject using the rAAVs described herein. In some embodiments, thedisclosure relates to a method for delivering a transgene to a subjectcomprising administering a rAAV to a subject, wherein the rAAVcomprises: (i) a capsid protein having a sequence selected from SEQ IDNOs: 19-27, and (ii) at least one transgene, and in which the rAAVinfects cells of a target tissue of the subject. In some embodiments,the at least one transgene encodes a protein (e.g., a therapeuticprotein). In some embodiments, the protein is an immunoglobulin heavychain or light chain or fragment thereof. In some embodiments, the atleast one transgene expresses a molecule that is involved in genomeediting (e.g., a component of a CRISPR-based genome editing system(e.g., a Cas9 or similar enzyme)).

In some embodiments, the at least one transgene encodes a smallinterfering nucleic acid. In some embodiments, the small interferingnucleic acid is a miRNA. In some embodiments, the small interferingnucleic acid is a miRNA sponge or TuD RNA that inhibits the activity ofat least one miRNA in the subject or animal. In some embodiments, themiRNA is expressed in a cell of the target tissue. In some embodiments,the target tissue is skeletal muscle, heart, liver, pancreas, brain orlung. In some embodiments, the transgene expresses a transcript thatcomprises at least one binding site for a miRNA, wherein the miRNAinhibits activity of the transgene, in a tissue other than the targettissue, by hybridizing to the binding site.

In some embodiments, the transgene comprises a tissue specific promoteror inducible promoter. In some embodiments, the tissue specific promoteris a liver-specific thyroxin binding globulin (TBG) promoter, an insulinpromoter, a glucagon promoter, a somatostatin promoter, a pancreaticpolypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatinekinase (MCK) promoter, a mammalian desmin (DES) promoter, a α-myosinheavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT) promoter.

In some embodiments, an rAAV disclosed herein is administered to asubject intravenously, intravascularly, transdermally, intraocularly,intrathecally, orally, intramuscularly, subcutaneously, intranasally, orby inhalation. In some embodiments, a subject is selected from a mouse,a rat, a rabbit, a dog, a cat, a sheep, a pig, and a non-human primate.In some embodiments, a subject is a human.

Also provided herein are isolated nucleic acids. In some embodiments,the disclosure relates to an isolated nucleic acid comprising a sequenceselected from the group consisting of: SEQ ID NOs: 8-18. In someembodiments, the disclosure relates to an isolated nucleic acid encodingan AAV capsid protein having an amino acid sequence selected from thegroup consisting of: SEQ ID NOs: 19-27. In some embodiments, thedisclosure relates to an isolated AAV capsid protein comprising an aminoacid sequence selected from the group consisting of: SEQ ID NOs: 19-27.

In some embodiments, the disclosure relates to a composition comprisingthe isolated AAV capsid proteins described herein. In some embodiments,the composition further comprises a pharmaceutically acceptable carrier.

In some embodiments, the disclosure relates to a kit for producing arAAV. In some embodiments, the kit comprises a container housing anisolated nucleic acid having a sequence of any one of SEQ ID NOs: 8-18.In some embodiments, the kit further comprises instructions forproducing the rAAV. In some embodiments, the kit further comprises atleast one container housing a recombinant AAV vector, wherein therecombinant AAV vector comprises a transgene.

In some embodiments, the disclosure relates to a kit comprising acontainer housing a recombinant AAV having an isolated AAV capsidprotein having an amino acid sequence as set forth in any of SEQ ID NOs:19-27.

Each of the features of the disclosure can encompass various embodimentsof the disclosure. It is, therefore, anticipated that each of thelimitations of the disclosure involving any one element or combinationsof elements can be included in each aspect of the disclosure. Thisdisclosure is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The disclosure iscapable of other embodiments and of being practiced or of being carriedout in various ways.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1A depicts a graphical representation of the AAV packaging strategyused in this example. VP1 and VP3 were expressed from one plasmid, whileVP2 containing an N-terminal Angiopep-2 (referred to here as BTP)insertion was expressed from a separate plasmid.

FIG. 1B depicts expression of capsid proteins VP1, VP2 and VP3 fromAAV9.47 and AAVrh8. Western blot analysis demonstrates successfulexpression of VP2 capsid protein containing the Angiopep-2 (BTP) insertin both parent capsids (AAV9.47 and AAVrh8) and confirms correctstoichiometry of all three capsid proteins.

FIG. 2 depicts increased biodistribution of vectors containingAngiopep-2-grafted VP2 capsid proteins. Both Angiopep-2-grafted AAVvectors (AAV9.47, AAVrh8) transduced brain endothelium, glia andcortical neurons at exceptional high efficiency when compared to parentcapsid controls.

FIG. 3 depicts enhanced neural tropism of Angiopep-2-grafted AAVvectors. Control AAVrh8 vector transduced neurons in the visual cortexbut not the striatum or thalamic region. The Angipep-2-grafted AAVrh8vector transduced thalamic neurons and neurons in the visual cortex,while neurons in the striatum were transduced only by theAngiopep-2-grafted AAV9.47 vector. Both AAV vectors transduced brainendothelium, glia and cortical neurons at exceptionally high efficiency.

FIG. 4 depicts results showing that angiopep-2-grafted AAV vectors donot exhibit increased transduction of non-target peripheral tissues whencompared to parent capsid control vectors.

FIG. 5 depicts insertion of a 19-amino acid alanine string “A-string”(AS) in the N-terminal domain of the VP2 capsid protein of AAV9.47increases CNS tropism. AS-grafted rAAV9.47 transduces cortical andstriatal neurons in the brain at unprecedented efficiency, while motorneurons and interneurons were transduced in the spinal cord.

FIG. 6 depicts AS-grafted rAAV9.47 transduces the cerebrum and spinalcord more efficiently than AAV9 and non-grafted rAAV9.47. Significantlymore vector genomes were present in the cerebrum and spinal cord ofsamples transduced with AS-grafted rAAV9.47 than in control samplestransfected with AAV9.

FIG. 7 depicts high efficiency transduction by AS-grafted rAAV9.47 inCNS tissue does not have increased off-target effects. No significantdifference in vector genomes was observed between rAAV9.47-AS and AAV9in non-target peripheral tissues (liver, skeletal muscle, heart, lung,kidney, pancreas and spleen).

FIGS. 8A-8C show CNS transduction profile of AAV-AS vector aftervascular infusion in adult mice. FIG. 8A shows an overview of GFPdistribution in brain and spinal cord in AAV-AS and AAV9 injected mice(5×10¹¹ vg/mouse). Representative images of coronal brain sectionslocated in relation to bregma at +0.5 mm, −0.5 mm and −1.80 mm, andcervical spinal cord (left to right) are shown. FIG. 8B showstransduction of neuronal populations in different brain regions. Blackarrows indicate examples of GFP-positive neurons identified bymorphology. Bar=50 μm. FIG. 8C shows the phenotype of transduced cellswas identified by double immunofluorescence staining with antibodies toGFP, pan-neuronal marker NeuN or striatal medium spiny neuron markerDARPP32. Neuronal transduction in spinal cord was examined in sectionsstained for GFP and NeuN. The large size and morphology of GFP-positiveneurons in the ventral spinal cord suggest a motor neuron identity.White arrows indicate examples of GFP-positive neurons. Bar=10 μm.

FIGS. 9A-9F show quantitative assessment of AAV-AS CNS transductionefficacy. FIG. 9A shows Western blot analysis of capsid proteincomposition of AAV vectors (1×10¹⁰ vg/lane) showed the presence of VP1,VP2 and VP3 capsid proteins. The poly-alanine VP2 fusion protein ofAAV-AS capsid (indicated by black arrow) has a higher molecular weightthan VP2 protein. FIG. 9B shows quantification of percentage ofGFP-positive neurons in striatum and thalamus of mice injected withAAV-AS-GFP or AAV9-GFP vectors. Data shown is mean±SD (n=4 biologicalreplicates per group). FIG. 9C shows AAV vector genome content incerebrum and spinal cords (n=4 animals per group). Age matchednon-injected mice were included as controls (not shown). FIG. 9D showsWestern blot analysis of GFP expression in cerebrum and spinal cord of 2animals per group. Signal intensity of GFP was normalized tocorresponding β-actin signal intensity for quantitative comparison. FIG.9E shows AAV vector genome content in liver and skeletal muscle(quadriceps) (n=4 animals per group). Data shown is mean±SD. FIG. 9Fshows Western blot analysis of GFP protein expression in liver andskeletal muscle (quadriceps). *p<0.05, **p<0.01, ***p<0.001,****p<0.0001 by Student's unpaired t-test.

FIGS. 10A-10B show neuronal transduction in cat after systemic deliveryof AAV-AS vectors. FIG. 10A shows transduction of neurons in the catbrain after systemic delivery of AAV-AS vector (1.29×10¹³ vg).Representative images (left) show GFP-positive cells with neuronalmorphology in various structures in the brain and spinal cord. Bar=50μm. FIG. 10B shows double immunofluorescence staining for GFP and NeuN(right) confirm the neuronal identity of GFP-positive cells in brain andspinal cord. White arrows indicate examples of GFP-positive neurons.Bar=50 μm.

FIGS. 11A-11B show Htt knockdown in mice upon intravenous administrationof AAV-AS-miR^(Htt) vector. FIG. 11A shows changes in Htt mRNA levels inbrain structures, cervical spinal cord and liver in wild type miceinjected systemically with AAV-AS or AAV9 vectors (n=4 per group)(9.4×10¹¹ vg/mouse) encoding a U6 promoter-driven artificial microRNA(miR^(Htt)) targeting mouse huntingtin mRNA. Values for each region werenormalized to Htt mRNA levels in age-matched PBS-injected mice. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001 by Student's unpaired t-test. Datashown is mean±error. FIG. 11B shows Western blot analysis of Htt and GFPprotein levels in brain structures, cervical spinal cord and liver ofthe same AAV-injected mice and PBS-injected controls. *p<0.05, **p<0.01,***p<0.001, ****p<0.0001 by Student's unpaired t-test. Data shown ismean±SD.

FIGS. 12A-12B show the biodistribution profile of AAV9.47. FIG. 12Ashows AAV vector genome content in cerebrum, spinal cord, liver, andmuscle of mice intravenously injected with AAV9.47-GFP or AAV9-GFPvectors (5×10¹¹ vg/mouse)(n=4 animals per group). FIG. 12B showsquantification of GFP-positive neurons per high power field in striatumand thalamus of injected mice (n=4 biological replicates per group).Data shown as mean±SD. **p<0.01 by Student's unpaired t-test.

FIGS. 13A-13B show construction of peptide-inserted vectors. FIG. 13Ashows a schematic diagram of VP2 capsid protein showing insertion siteof peptide and G₄S linker. FIG. 13B shows an illustration of packagingstrategy. VP1 and VP3 are expressed separately (top) from VP2 fused withpeptide (below).

FIG. 14 shows transduction profile of AAV-AS and AAV9 vectors acrossmultiple CNS regions after systemic delivery. Bar=50 μm.

FIG. 15 shows cell binding studies of native and peptide-modified AAVvector. No significant difference in binding to Lec2 or ProS cells. Datashown as mean±SD. Experiment was performed with N=3 biologicalreplicates.

FIGS. 16A-16C show the brain transduction profile of AAVrh8-AS vector.Distribution of GFP positive cells was assess in immunostainedhistological sections. FIG. 16A shows Western blot analysis of AAVrh8and AAVrh8-AS vectors (1×10¹⁰ vg total) for the presence of capsidproteins VP1, 2 and 3. Arrow indicates the position of the AS-VP2 fusionprotein on the blot. FIG. 16B and 16C show transduced cell distributionin brain. Sections represented in FIG. 16B correspond to coronal planes+0.5 mm and −1.80 mm from the bregma plane. Bar=50 μm.

FIGS. 17A-17B show biodistribution of AAVrh8-AS and AAVrh8 vectors.Quantification of AAV vector genome content in (FIG. 17A) different CNSregions or (FIG. 17B) liver. Data shown as mean±SD. Experiment wasperformed with N=3 per group. *p<0.05, **p<0.01, ****p<0.0001 byStudent's unpaired t-test.

DETAILED DESCRIPTION

In some embodiments, recombinant AAVs (rAAVs) are provided herein thathave distinct tissue targeting properties that make them useful forcertain gene therapy and research applications. AAV capsid proteins areprovided herein that comprise heterologous targeting peptides thatconfer desired cell and/or tissue targeting properties on rAAVscomprising such capsid proteins. For examples, in some aspects, aheterologous targeting peptide is grafted in or to a capsid protein ofan rAAV that facilitates transport of the rAAV across the blood brainbarrier (BBB). Accordingly, in some aspects, rAAV-based methods fordelivering a transgene to a target tissue in a subject are provided. Thetransgene delivery methods may be used for gene therapy (e.g., to treatdisease) or research (e.g., to create a somatic transgenic animal model)applications.

Isolated AAV Capsid Proteins and Nucleic Acids Encoding the Same

AAVs that infect mammals, particularly non-human primates, are usefulfor creating gene transfer vectors for clinical development and humangene therapy applications. The disclosure provides in some aspects novelAAV capsid proteins developed through functional mutagenesis. In someembodiments, an AAV capsid is provided that has an amino acid sequenceselected from the group consisting of SEQ ID NO:19-27. In someembodiments, an AAV capsid is provided that is encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NO: 8-18.

An example of an isolated nucleic acid that encodes an AAV capsidprotein is a nucleic acid having a sequence selected from the groupconsisting of: SEQ ID NO: 8-18 as well as nucleic acids havingsubstantial homology thereto. In some embodiments, isolated nucleicacids that encode AAV capsids are provided that have sequences selectedfrom: SEQ ID NO:8-18.

In some embodiments, nucleic acids are provided that encode an AAVcapsid having a peptide grafted within its capsid sequence (e.g., a AAV9capsid) and up to 5, up to 10, up to 20, up to 30, up to 40, up to 50,up to 100 other amino acid alternations.

In some embodiments, a fragment (portion) of an isolated nucleic acidencoding a AAV capsid sequence may be useful for constructing a nucleicacid encoding a desired capsid sequence. Fragments may be of anyappropriate length (e.g., at least 9, at least 18, at least 36, at least72, at least 144, at least 288, at least 576, at least 1152 or morenucleotides in length). For example, a fragment of nucleic acid sequenceencoding a variant amino acid (compared with a known AAV serotype) maybe used to construct, or may be incorporated within, a nucleic acidsequence encoding an AAV capsid sequence to alter the properties of theAAV capsid. For example, a nucleic sequence encoding an AAV variant maycomprise n amino acid variants (e.g., in which n=1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more) compared with a known AAV serotype (e.g., AAV9). Arecombinant cap sequence may be constructed having one or more of the namino acid variants by incorporating fragments of a nucleic acidsequence comprising a region encoding a variant amino acid into thesequence of a nucleic acid encoding the known AAV serotype. Thefragments may be incorporated by any appropriate method, including usingsite directed mutagenesis. In some embodiments, polypeptide fragmentsthat are not normally present in AAV capsid proteins may be incorporatedinto a recombinant cap sequence. In some embodiments, the polypeptidefragment is grafted onto the recombinant cap sequence. Thus, new AAVvariants may be created having new properties.

As used herein, “grafting” refers to joining or uniting of one moleculewith another molecule. In some embodiments, the term grafting refers tojoining or uniting of at least two molecules such that one of the atleast two molecules is inserted within another of the at least twomolecules. In some embodiments, the term grafting refers to joining oruniting of at least two polymeric molecules such that one of the atleast two molecules is appended to another of the at least twomolecules. In some embodiments, the term grafting refers to joining oruniting of one polymeric molecule (e.g., a nucleic acid, a polypeptide)with another polymeric molecule (e.g., a nucleic acid, a polypeptide).In some embodiments, the term grafting refers to joining or uniting ofat least two nucleic acid molecules such that one of the at least twonucleic acid molecules is inserted within another of the at least twonucleic acid molecules. In some embodiments, the term grafting refers tojoining or uniting of at least two nucleic acid molecules such that oneof the at least two molecules is appended to another of the at least twonucleic acid molecules.

In some embodiments, a nucleic acid formed through grafting (a graftednucleic acid) encodes a chimeric protein. In some embodiments, a graftednucleic acid encodes a chimeric protein, such that one polypeptide iseffectively inserted into another polypeptide (e.g., not directlyconjugated before the N-terminus or after the C-terminus), therebycreating a contiguous fusion of two polypeptides. In some embodiments, agrafted nucleic acid encodes a chimeric protein, such that onepolypeptide is effectively appended to another polypeptide (e.g.,directly conjugated before the N-terminus or after the C-terminus),thereby creating a contiguous fusion of two polypeptides. In someembodiments, the term grafting refers to joining or uniting of at leasttwo polypeptides, or fragments thereof, such that one of the at leasttwo polypeptides or fragments thereof is inserted within another of theat least two polypeptides or fragments thereof. In some embodiments, theterm grafting refers to joining or uniting of at least two polypeptidesor fragments thereof such that one of the at least two polypeptides orfragments thereof is appended to another of the at least twopolypeptides or fragments thereof.

In some embodiments, the disclosure relates to an adeno-associated virus(AAV) capsid protein comprising a AAV capsid protein having anN-terminally grafted heterologous targeting peptide in a range of 11 to31 amino acids in length. In some embodiments, the disclosure relates toan adeno-associated virus (AAV) capsid protein comprising a AAV capsidprotein having an N-terminally grafted heterologous targeting peptide ina range of 11 to 27 amino acids in length. In some embodiments, theheterologous targeting peptide is in a range of 17 to 21 amino acids inlength. In some embodiments, the heterologous targeting peptide is 19amino acids in length. In some embodiments, the heterologous targetingpeptide is a polypeptide represented by SEQ ID NO: 5. In someembodiments, the heterologous targeting peptide is represented by SEQ IDNO: 7. In some embodiments, the heterologous targeting peptide is apolypeptide encoded by the nucleic acid sequence represented by SEQ IDNO: 29.

In some embodiments, a heterologous targeting protein further comprisesan N-terminal tag, for example a polypeptide tag. As used herein,“N-terminal tag” refers to a peptide sequence that is covalently linked(e.g., grafted) onto the N-terminus of a recombinant protein. AnN-terminal tag can be directly linked to a recombinant protein (e.g.,contiguously linked) or indirectly linked to a recombinant protein(e.g., via a linker sequence). Peptide tags may include, for example,Myc tag, His tag, FLAG tag, chitin binding protein (CBP) tag, maltosebinding protein tag (MBP), and human influenza hemagglutinin (HA) tag,and glutathione-S-transferase (GST) tag. Additional descriptions ofprotein tags can be found, for example, in Lichty et al., Protein Expr.Purif., 41(1): 98-105 (2005) the pertinent contents of which areincorporated herein by reference.

In some embodiments, a heterologous targeting peptide comprises anN-terminal Myc tag. In some embodiments, a heterologous targetingpeptide comprises an N-terminal poly-histidine tag (His) tag. In someembodiments, the His tag ranges from about 6 amino acids to about 10amino acids in length. In some embodiments, the His tag is six aminoacids in length and is referred to as a hex-His tag. Without wishing tobe bound by any particular theory, the presence of an N-terminal proteintag on a heterologous targeting peptide may facilitate purification ofan AAV comprising the tagged heterologous targeting peptide.

In some embodiments, the AAV capsid protein further comprises a linker.In some embodiments, the linker is conjugated to the C-terminus of theN-terminally grafted heterologous targeting peptide. In someembodiments, the linker is conjugated to the N-terminus of theN-terminally grafted heterologous targeting peptide. In someembodiments, one linker is conjugated to the N-terminus of theN-terminally grafted heterologous targeting peptide and a second linkeris conjugated to the C-terminus of the N-terminally grafted heterologoustargeting peptide. In some embodiments, the linker is a glycine-richlinker. In some embodiments, the linker comprises at least two glycineresidues. In some embodiments, the polypeptide repeat comprises GGGGS(SEQ ID NO: 28). In some embodiments, the linker comprises a formulaselected from the group consisting of: [G]_(n), [G]_(n)S, [GS]_(n), and[GGSG]_(n), wherein G is glycine and wherein n is an integer greaterthan one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or more). In some embodiments, n is aninteger in a range of 2 to 10, 2 to 20, 5 to 10, 5 to 15, or 5 to 25.Accordingly, in some embodiments, a heterologous targeting peptides isconjugated toa linker. In some embodiments, heterologous targetingpeptide is provided with an N terminal methionine, reflecting the aminoacid residue corresponding to a start codon, for example. In someembodiments, peptides of the following peptide sequence are providedMAAAAAAAAAAAAAAAAAAAGGGGS (SEQ ID NO: 30); MAAAAAAAAAAAAAAAAAAA (SEQ IDNO: 31); and AAAAAAAAAAAAAAAAAAAGGGGS(SEQ ID NO: 32) and may be graftedwithin or to capsid proteins to alter targeting.

In some aspects, the disclosure relates to a recombinant AAV comprisingan AAV capsid protein having an N-terminally grafted heterologoustargeting peptide that mediates transcytosis across the blood-brainbarrier. In some embodiments, transport of molecules across the bloodbrain barrier is mediated by lipoprotein receptor-related proteins (LRP)or suitable epitopes derived therefrom. LPRs are members of a largeconserved family of endocytic receptors that bind and internalize abroad spectrum of ligands including but not limited to lipoproteins,proteinases, proteinase inhibitor complexes, viruses, bacterial toxinsand extracellular matrix proteins. Non-limiting examples of LRPs includeLRP1, LRP1B, LRP2 (megalin), LRP3, LRP4, LRPS, LRP6, LRP8(apolipoprotein e receptor), LRP10, and Angiopep-2 and suitablefragments thereof. In some embodiments, the N-terminally graftedheterologous targeting peptides disclosed herein are LRP ligands. Insome embodiments, the N-terminally grafted heterologous targetingpeptide is Angiopep-2. In some embodiments, the N-terminally graftedheterologous targeting peptide is a polypeptide represented by SEQ IDNO:5. In some embodiments, the N-terminally grafted heterologoustargeting peptide is encoded by the nucleic acid represented in SEQ IDNO:29. Other N-terminally grafted heterologous targeting peptides thatmediate transcytosis across the blood-brain barrier are alsocontemplated herein.

In some embodiments, the N-terminally grafted heterologous targetingpeptide is a polypeptide represented by SEQ ID NO: 7. In someembodiments, the N-terminally grafted heterologous targeting peptide isa polypeptide represented by the [A]_(n), wherein A is alanine and n isan integer greater than 5 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more).In some embodiments, n is an integer in a range of 5 to 10, 5 to 11, 5to 12, 5 to 13, 5 to 14, 5 to 15, 5 to 20, 5 to 25, 15 to 25, 15 to 30,17 to 21 or 18 to 20.

In some cases, fragments of capsid proteins disclosed herein areprovided. Such fragments may be at least 10, at least 20, at least 50,at least 100, at least 200, at least 300, at least 400, at least 500 ormore amino acids in length. In some embodiments, chimeric capsidproteins are provided that comprise one or more fragments of one or morecapsid proteins disclosed herein.

“Homology” refers to the percent identity between two polynucleotides ortwo polypeptide moieties. The term “substantial homology”, whenreferring to a nucleic acid, or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in about 90 to 100% of the alignedsequences. When referring to a polypeptide, or fragment thereof, theterm “substantial homology” indicates that, when optimally aligned withappropriate gaps, insertions or deletions with another polypeptide,there is nucleotide sequence identity in about 90 to 100% of the alignedsequences. The term “highly conserved” means at least 80% identity,preferably at least 90% identity, and more preferably, over 97%identity. In some cases, highly conserved may refer to 100% identity.Identity is readily determined by one of skill in the art by, forexample, the use of algorithms and computer programs known by those ofskill in the art.

As described herein, alignments between sequences of nucleic acids orpolypeptides are performed using any of a variety of publicly orcommercially available Multiple Sequence Alignment Programs, such as“Clustal W”, accessible through Web Servers on the internet.Alternatively, Vector NTI utilities may also be used. There are also anumber of algorithms known in the art which can be used to measurenucleotide sequence identity, including those contained in the programsdescribed above. As another example, polynucleotide sequences can becompared using BLASTN, which provides alignments and percent sequenceidentity of the regions of the best overlap between the query and searchsequences. Similar programs are available for the comparison of aminoacid sequences, e.g., the “Clustal X” program, BLASTP. Typically, any ofthese programs are used at default settings, although one of skill inthe art can alter these settings as needed. Alternatively, one of skillin the art can utilize another algorithm or computer program whichprovides at least the level of identity or alignment as that provided bythe referenced algorithms and programs. Alignments may be used toidentify corresponding amino acids between two proteins or peptides. A“corresponding amino acid” is an amino acid of a protein or peptidesequence that has been aligned with an amino acid of another protein orpeptide sequence. Corresponding amino acids may be identical ornon-identical. A corresponding amino acid that is a non-identical aminoacid may be referred to as a variant amino acid.

Alternatively for nucleic acids, homology can be determined byhybridization of polynucleotides under conditions which form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s), and size determination of thedigested fragments. DNA sequences that are substantially homologous canbe identified in a Southern hybridization experiment under, for example,stringent conditions, as defined for that particular system. Definingappropriate hybridization conditions is within the skill of the art.

A “nucleic acid” sequence refers to a DNA or RNA sequence. In someembodiments, proteins and nucleic acids of the disclosure are isolated.As used herein, the term “isolated” means artificially produced. As usedherein with respect to nucleic acids, the term “isolated” means: (i)amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art. As used herein with respect to proteins or peptides, the term“isolated” refers to a protein or peptide that has been isolated fromits natural environment or artificially produced (e.g., by chemicalsynthesis, by recombinant DNA technology, etc.).

The skilled artisan will also realize that conservative amino acidsubstitutions may be made to provide functionally equivalent variants,or homologs of the capsid proteins. In some aspects the disclosureembraces sequence alterations that result in conservative amino acidsubstitutions. As used herein, a conservative amino acid substitutionrefers to an amino acid substitution that does not alter the relativecharge or size characteristics of the protein in which the amino acidsubstitution is made. Variants can be prepared according to methods foraltering polypeptide sequence known to one of ordinary skill in the artsuch as are found in references that compile such methods, e.g.,Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,New York, 1989, or Current Protocols in Molecular Biology, F. M.Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservativesubstitutions of amino acids include substitutions made among aminoacids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K,R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Therefore, one canmake conservative amino acid substitutions to the amino acid sequence ofthe proteins and polypeptides disclosed herein.

Furthermore, nucleic acids can be tailored for optimal gene expressionbased on optimization of nucleotide sequence to reflect the codon biasof a host cell. The skilled artisan appreciates that gene expression maybe improved if codon usage is biased towards those codons favored by thehost.

Recombinant AAVs

In some aspects, the disclosure provides isolated AAVs. As used hereinwith respect to AAVs, the term “isolated” refers to an AAV that has beenartificially obtained or produced. Isolated AAVs may be produced usingrecombinant methods. Such AAVs are referred to herein as “recombinantAAVs”. Recombinant AAVs (rAAVs) preferably have tissue-specifictargeting capabilities, such that a transgene of the rAAV will bedelivered specifically to one or more predetermined tissue(s). The AAVcapsid is an important element in determining these tissue-specifictargeting capabilities. Thus, an rAAV having a capsid appropriate forthe tissue being targeted can be selected. In some embodiments, the rAAVcomprises a capsid protein having an amino acid sequence as set forth inany one of SEQ ID NOs 19-27, or a protein having substantial homologythereto.

In some embodiments, the rAAVs of the disclosure are pseudotyped rAAVs.Pseudotyping is the process of producing viruses or viral vectors incombination with foreign viral envelope proteins. The result is apseudotyped virus particle. With this method, the foreign viral envelopeproteins can be used to alter host tropism or an increased/decreasedstability of the virus particles. In some aspects, a pseudotyped rAAVcomprises nucleic acids from two or more different AAVs, wherein thenucleic acid from one AAV encodes a capsid protein and the nucleic acidof at least one other AAV encodes other viral proteins and/or the viralgenome. In some embodiments, a pseudotyped rAAV refers to an AAVcomprising an inverted terminal repeats (ITRs) of one AAV serotype andan capsid protein of a different AAV serotype. For example, apseudotyped AAV vector containing the ITRs of serotype X encapsidatedwith the proteins of Y will be designated as AAVX/Y (e.g., AAV2/1 hasthe ITRs of AAV2 and the capsid of AAV1). In some embodiments,pseudotyped rAAVs may be useful for combining the tissue-specifictargeting capabilities of a capsid protein from one AAV serotype withthe viral DNA from another AAV serotype, thereby allowing targeteddelivery of a transgene to a target tissue.

Methods for obtaining recombinant AAVs having a desired capsid proteinare well known in the art. (See, for example, US Patent ApplicationPublication Number US 2003/0138772, the contents of which areincorporated herein by reference in their entirety). Typically themethods involve culturing a host cell which contains a nucleic acidsequence encoding an AAV capsid protein (e.g., a nucleic acid having asequence as set forth in any one of SEQ ID NOs 8-18 or fragment thereof;a functional rep gene; a recombinant AAV vector composed of, AAVinverted terminal repeats (ITRs) and a transgene; and sufficient helperfunctions to permit packaging of the recombinant AAV vector into the AAVcapsid proteins. In some embodiments, capsid proteins are structuralproteins endocoded by the cap gene of an AAV. In some embodiments, AAVscomprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2and VP3), all of which are transcribed from a single cap gene viaalternative splicing. In some embodiments, the molecular weights of VP1,VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62kDa. In some embodiments, upon translation, capsid proteins form aspherical 60-mer protein shell around the viral genome. In someembodiments, capsid proteins protect a viral genome, deliver a genomeand/or interact with a host cell. In some aspects, capsid proteinsdeliver the viral genome to a host in a tissue specific manner. In someembodiments, an N-terminally grafted heterologous targeting peptide ispresent on all three capsid proteins (e.g., VP1, VP2, VP3) of a rAAV. Insome embodiments, an N-terminally grafted heterologous targeting peptideis present on two of the capsid proteins (e.g., VP2 and VP3) of a rAAV.In some embodiments, an N-terminally grafted heterologous targetingpeptide is present on a single capsid protein of a rAAV. In someembodiments, an N-terminally grafted heterologous targeting peptide ispresent on the VP2 capsid protein of the rAAV.

In some embodiments, the disclosure relates to an adeno-associated virus(AAV) capsid protein comprising: an AAV capsid protein having anN-terminally grafted heterologous targeting peptide, wherein the AAVcapsid protein is not of an AAV2 serotype. In some embodiments, the AAVcapsid protein is of an AAV serotype selected from the group consistingof AAV3, AAV4, AAV5, AAV6, AAV8, AAVrh8 AAV9, and AAV10. In someembodiments, the capsid protein having an N-terminally graftedheterologous targeting peptide is a viral protein 2 (VP2) capsidprotein. In some embodiments, the AAV capsid protein having anN-terminally grafted heterologous targeting peptide is of a serotypederived from a non-human primate. In some embodiments, the AAV capsidprotein having an N-terminally grafted heterologous targeting peptide isof a AAVrh8 serotype. In some embodiments, the AAV capsid protein havingan N-terminally grafted heterologous targeting peptide is of an AAV9,optionally AAV9.47, serotype.

In some aspects, the disclosure relates to the location within an AAVcapsid protein where a heterologous targeting peptide is grafted. Insome embodiments, an heterologous targeting peptide is N-terminallygrafted. In some embodiments, an N-terminally grafted heterologoustargeting peptide is inserted before the fifth, fourth or third aminoacid after the N-terminal amino acid of the capsid protein. In someembodiments, an N-terminally grafted heterologous targeting peptide isinserted before the second N-terminal amino acid of the capsid protein.In some embodiments, an N-terminally grafted heterologous targetingpeptide is inserted after the first N-terminal amino acid of the capsidprotein. In some embodiments, an N-terminally grafted heterologoustargeting peptide is inserted after the first N-terminal methionineresidue of the capsid protein.

In some embodiments, components to be cultured in the host cell topackage a rAAV vector in an AAV capsid may be provided to the host cellin trans. Alternatively, any one or more of the required components(e.g., recombinant AAV vector, rep sequences, cap sequences, and/orhelper functions) may be provided by a stable host cell which has beenengineered to contain one or more of the required components usingmethods known to those of skill in the art. Most suitably, such a stablehost cell will contain the required component(s) under the control of aninducible promoter. However, the required component(s) may be under thecontrol of a constitutive promoter. Examples of suitable inducible andconstitutive promoters are provided herein, in the discussion ofregulatory elements suitable for use with the transgene. In stillanother alternative, a selected stable host cell may contain selectedcomponent(s) under the control of a constitutive promoter and otherselected component(s) under the control of one or more induciblepromoters. For example, a stable host cell may be generated which isderived from 293 cells (which contain E1 helper functions under thecontrol of a constitutive promoter), but which contain the rep and/orcap proteins under the control of inducible promoters. Still otherstable host cells may be generated by one of skill in the art.

In some embodiments, the disclosure relates to a host cell containing anucleic acid that comprises a coding sequence selected from the groupconsisting of: SEQ ID NOs: 8-18 that is operably linked to a promoter.In some embodiments, the disclosure relates to a composition comprisingthe host cell described above. In some embodiments, the compositioncomprising the host cell above further comprises a cryopreservative.

The recombinant AAV vector, rep sequences, cap sequences, and helperfunctions useful for producing the rAAV of the disclosure may bedelivered to the packaging host cell using any appropriate geneticelement (vector). The selected genetic element may be delivered by anysuitable method, including those described herein. The methods used toconstruct any embodiment of this disclosure are known to those withskill in nucleic acid manipulation and include genetic engineering,recombinant engineering, and synthetic techniques. See, e.g., Sambrooket al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virionsare well known and the selection of a suitable method is not alimitation on the present disclosure. See, e.g., K. Fisher et al, J.Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

In some embodiments, recombinant AAVs may be produced using the tripletransfection method (described in detail in U.S. Pat. No. 6,001,650).Typically, the recombinant AAVs are produced by transfecting a host cellwith an recombinant AAV vector (comprising a transgene) to be packagedinto AAV particles, an AAV helper function vector, and an accessoryfunction vector. An AAV helper function vector encodes the “AAV helperfunction” sequences (i.e., rep and cap), which function in trans forproductive AAV replication and encapsidation. Preferably, the AAV helperfunction vector supports efficient AAV vector production withoutgenerating any detectable wild-type AAV virions (i.e., AAV virionscontaining functional rep and cap genes). Non-limiting examples ofvectors suitable for use with the present disclosure include pHLP19,described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described inU.S. Pat. No. 6,156,303, the entirety of both incorporated by referenceherein. The accessory function vector encodes nucleotide sequences fornon-AAV derived viral and/or cellular functions upon which AAV isdependent for replication (i.e., “accessory functions”). The accessoryfunctions include those functions required for AAV replication,including, without limitation, those moieties involved in activation ofAAV gene transcription, stage specific AAV mRNA splicing, AAV DNAreplication, synthesis of cap expression products, and AAV capsidassembly. Viral-based accessory functions can be derived from any of theknown helper viruses such as adenovirus, herpesvirus (other than herpessimplex virus type-1), and vaccinia virus.

In some aspects, the disclosure provides transfected host cells. Theterm “transfection” is used to refer to the uptake of foreign DNA by acell, and a cell has been “transfected” when exogenous DNA has beenintroduced through the cell membrane. A number of transfectiontechniques are generally known in the art. See, e.g., Graham et al.(1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, alaboratory manual, Cold Spring Harbor Laboratories, New York, Davis etal. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al.(1981) Gene 13:197. Such techniques can be used to introduce one or moreexogenous nucleic acids, such as a nucleotide integration vector andother nucleic acid molecules, into suitable host cells.

A “host cell” refers to any cell that harbors, or is capable ofharboring, a substance of interest. Often a host cell is a mammaliancell. A host cell may be used as a recipient of an AAV helper construct,an AAV minigene plasmid, an accessory function vector, or other transferDNA associated with the production of recombinant AAVs. The termincludes the progeny of the original cell which has been transfected.Thus, a “host cell” as used herein may refer to a cell which has beentransfected with an exogenous DNA sequence. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

As used herein, the term “cell line” refers to a population of cellscapable of continuous or prolonged growth and division in vitro. Often,cell lines are clonal populations derived from a single progenitor cell.It is further known in the art that spontaneous or induced changes canoccur in karyotype during storage or transfer of such clonalpopulations. Therefore, cells derived from the cell line referred to maynot be precisely identical to the ancestral cells or cultures, and thecell line referred to includes such variants.

As used herein, the terms “recombinant cell” refers to a cell into whichan exogenous DNA segment, such as DNA segment that leads to thetranscription of a biologically-active polypeptide or production of abiologically active nucleic acid such as an RNA, has been introduced.

As used herein, the term “vector” includes any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, artificial chromosome,virus, virion, etc., which is capable of replication when associatedwith the proper control elements and which can transfer gene sequencesbetween cells. Thus, the term includes cloning and expression vehicles,as well as viral vectors. In some embodiments, useful vectors arecontemplated to be those vectors in which the nucleic acid segment to betranscribed is positioned under the transcriptional control of apromoter. A “promoter” refers to a DNA sequence recognized by thesynthetic machinery of the cell, or introduced synthetic machinery,required to initiate the specific transcription of a gene. The phrases“operatively positioned,” “under control” or “under transcriptionalcontrol” means that the promoter is in the correct location andorientation in relation to the nucleic acid to control RNA polymeraseinitiation and expression of the gene. The term “expression vector orconstruct” means any type of genetic construct containing a nucleic acidin which part or all of the nucleic acid encoding sequence is capable ofbeing transcribed. In some embodiments, expression includestranscription of the nucleic acid, for example, to generate abiologically-active polypeptide product or inhibitory RNA (e.g., shRNA,miRNA, miRNA inhibitor) from a transcribed gene.

The foregoing methods for packaging recombinant vectors in desired AAVcapsids to produce the rAAVs of the disclosure are not meant to belimiting and other suitable methods will be apparent to the skilledartisan.

Recombinant AAV Vectors

“Recombinant AAV (rAAV) vectors” of the disclosure are typicallycomposed of, at a minimum, a transgene and its regulatory sequences, and5′ and 3′ AAV inverted terminal repeats (ITRs). It is this recombinantAAV vector which is packaged into a capsid protein and delivered to aselected target cell. In some embodiments, the transgene is a nucleicacid sequence, heterologous to the vector sequences, which encodes apolypeptide, protein, functional RNA molecule (e.g., miRNA, miRNAinhibitor) or other gene product, of interest. The nucleic acid codingsequence is operatively linked to regulatory components in a mannerwhich permits transgene transcription, translation, and/or expression ina cell of a target tissue.

In some embodiments, the disclosure relates to a recombinant AAV (rAAV)comprising a capsid protein having an N-terminally grafted heterologoustargeting peptide, wherein the N-terminally grafted heterologoustargeting peptide is present only in the VP2 capsid protein. In someembodiments, the rAAV comprises a capsid protein having an amino acidsequence selected from the group consisting of: SEQ ID NOs: 19-27.

The AAV sequences of the vector typically comprise the cis-acting 5′ and3′ inverted terminal repeat sequences (See, e.g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168(1990)). The ITR sequences are about 145 bp in length. Preferably,substantially the entire sequences encoding the ITRs are used in themolecule, although some degree of minor modification of these sequencesis permissible. The ability to modify these ITR sequences is within theskill of the art. (See, e.g., texts such as Sambrook et al, “MolecularCloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory,New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). Anexample of such a molecule employed in the present disclosure is a“cis-acting” plasmid containing the transgene, in which the selectedtransgene sequence and associated regulatory elements are flanked by the5′ and 3′ AAV ITR sequences. The AAV ITR sequences may be obtained fromany known AAV, including presently identified mammalian AAV types.

In some embodiments, the rAAVs of the disclosure are pseudotyped rAAVs.For example, a pseudotyped AAV vector containing the ITRs of serotype Xencapsidated with the proteins of Y will be designated as AAVX/Y (e.g.,AAV2/1 has the ITRs of AAV2 and the capsid of AAV1). In someembodiments, pseudotyped rAAVs may be useful for combining thetissue-specific targeting capabilities of a capsid protein from one AAVserotype with the viral DNA from another AAV serotype, thereby allowingtargeted delivery of a transgene to a target tissue.

In addition to the major elements identified above for the recombinantAAV vector, the vector also includes conventional control elementsnecessary which are operably linked to the transgene in a manner whichpermits its transcription, translation and/or expression in a celltransfected with the plasmid vector or infected with the virus producedby the disclosure. As used herein, “operably linked” sequences includeboth expression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance to control the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters which are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

As used herein, a nucleic acid sequence (e.g., coding sequence) andregulatory sequences are said to be “operably” linked when they arecovalently linked in such a way as to place the expression ortranscription of the nucleic acid sequence under the influence orcontrol of the regulatory sequences. If it is desired that the nucleicacid sequences be translated into a functional protein, two DNAsequences are said to be operably linked if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably linked to a nucleic acidsequence if the promoter region were capable of effecting transcriptionof that DNA sequence such that the resulting transcript might betranslated into the desired protein or polypeptide. Similarly two ormore coding regions are operably linked when they are linked in such away that their transcription from a common promoter results in theexpression of two or more proteins having been translated in frame. Insome embodiments, operably linked coding sequences yield a fusionprotein. In some embodiments, operably linked coding sequences yield afunctional RNA (e.g., shRNA, miRNA, miRNA inhibitor).

For nucleic acids encoding proteins, a polyadenylation sequencegenerally is inserted following the transgene sequences and before the3′ AAV ITR sequence. A rAAV construct useful in the present disclosuremay also contain an intron, desirably located between thepromoter/enhancer sequence and the transgene. One possible intronsequence is derived from SV-40, and is referred to as the SV-40 T intronsequence. Another vector element that may be used is an internalribosome entry site (IRES). An IRES sequence is used to produce morethan one polypeptide from a single gene transcript. An IRES sequencewould be used to produce a protein that contain more than onepolypeptide chains. Selection of these and other common vector elementsare conventional and many such sequences are available [see, e.g.,Sambrook et al, and references cited therein at, for example, pages 3.183.26 and 16.17 16.27 and Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, 1989]. In some embodiments, a Footand Mouth Disease Virus 2A sequence is included in polyprotein; this isa small peptide (approximately 18 amino acids in length) that has beenshown to mediate the cleavage of polyproteins (Ryan, M D et al., EMBO,1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996; p.8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin,C et al., The Plant Journal, 1999; 4: 453-459). The cleavage activity ofthe 2A sequence has previously been demonstrated in artificial systemsincluding plasmids and gene therapy vectors (AAV and retroviruses)(Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M et al., JVirology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy,2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4:453-459; de Felipe, P et al., Gene Therapy, 1999; 6: 198-208; de Felipe,P et al., Human Gene Therapy, 2000; 11: 1921-1931.; and Klump, H et al.,Gene Therapy, 2001; 8: 811-817).

The precise nature of the regulatory sequences needed for geneexpression in host cells may vary between species, tissues or celltypes, but shall in general include, as necessary, 5′ non-transcribedand 5′ non-translated sequences involved with the initiation oftranscription and translation respectively, such as a TATA box, cappingsequence, CAAT sequence, enhancer elements, and the like. Especially,such 5′ non-transcribed regulatory sequences will include a promoterregion that includes a promoter sequence for transcriptional control ofthe operably joined gene. Regulatory sequences may also include enhancersequences or upstream activator sequences as desired. The vectors of thedisclosure may optionally include 5′ leader or signal sequences. Thechoice and design of an appropriate vector is within the ability anddiscretion of one of ordinary skill in the art.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], theSV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EFlapromoter [Invitrogen].

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art.Examples of inducible promoters regulated by exogenously suppliedpromoters include the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); theecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA,93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al,Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), thetetracycline-inducible system (Gossen et al, Science, 268:1766-1769(1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518(1998)), the RU486-inducible system (Wang et al, Nat. Biotech.,15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)) and therapamycin-inducible system (Magari et al, J. Clin. Invest.,100:2865-2872 (1997)). Still other types of inducible promoters whichmay be useful in this context are those which are regulated by aspecific physiological state, e.g., temperature, acute phase, aparticular differentiation state of the cell, or in replicating cellsonly.

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

In some embodiments, the regulatory sequences impart tissue-specificgene expression capabilities. In some cases, the tissue-specificregulatory sequences bind tissue-specific transcription factors thatinduce transcription in a tissue specific manner. Such tissue-specificregulatory sequences (e.g., promoters, enhancers, etc.) are well knownin the art. Exemplary tissue-specific regulatory sequences include, butare not limited to the following tissue specific promoters: aliver-specific thyroxin binding globulin (TBG) promoter, an insulinpromoter, a glucagon promoter, a somatostatin promoter, a pancreaticpolypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatinekinase (MCK) promoter, a mammalian desmin (DES) promoter, a a-myosinheavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT) promoter.Other exemplary promoters include Beta-actin promoter, hepatitis B viruscore promoter, Sandig et al., Gene Ther., 3:1002-9 (1996);alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther.,7:1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol. Biol.Rep., 24:185-96 (1997)); bone sialoprotein promoter (Chen et al., J.Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J.Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cellreceptor α-chain promoter, neuronal such as neuron-specific enolase(NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15(1993)), neurofilament light-chain gene promoter (Piccioli et al., Proc.Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgfgene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), among otherswhich will be apparent to the skilled artisan.

In some embodiments, one or more binding sites for one or more of miRNAsare incorporated in a transgene of a rAAV vector, to inhibit theexpression of the transgene in one or more tissues of a subjectharboring the transgene. The skilled artisan will appreciate thatbinding sites may be selected to control the expression of a transgenein a tissue specific manner. For example, binding sites for theliver-specific miR-122 may be incorporated into a transgene to inhibitexpression of that transgene in the liver. The target sites in the mRNAmay be in the 5′ UTR, the 3′ UTR or in the coding region. Typically, thetarget site is in the 3′ UTR of the mRNA. Furthermore, the transgene maybe designed such that multiple miRNAs regulate the mRNA by recognizingthe same or multiple sites. The presence of multiple miRNA binding sitesmay result in the cooperative action of multiple RISCs and providehighly efficient inhibition of expression. The target site sequence maycomprise a total of 5-100, 10-60, or more nucleotides. The target sitesequence may comprise at least 5 nucleotides of the sequence of a targetgene binding site.

Recombinant AAV Vector: Transgene Coding Sequences

The composition of the transgene sequence of the rAAV vector will dependupon the use to which the resulting vector will be put. For example, onetype of transgene sequence includes a reporter sequence, which uponexpression produces a detectable signal. In another example, thetransgene encodes a therapeutic protein or therapeutic functional RNA.In another example, the transgene encodes a protein or functional RNAthat is intended to be used for research purposes, e.g., to create asomatic transgenic animal model harboring the transgene, e.g., to studythe function of the transgene product. In another example, the transgeneencodes a protein or functional RNA that is intended to be used tocreate an animal model of disease. Appropriate transgene codingsequences will be apparent to the skilled artisan.

Also contemplated herein are methods of delivering a transgene to asubject using the rAAVs described herein. In some embodiments, thedisclosure relates to a method for delivering a transgene to a subjectcomprising administering a rAAV to a subject, wherein the rAAVcomprises: (i) a capsid protein having a sequence selected from SEQ IDNOs: 19-27, and (ii) at least one transgene, and wherein the rAAVinfects cells of a target tissue of the subject. In some embodiments ofthe method, at least one transgene encodes a protein. In someembodiments, the protein is an immunoglobulin heavy chain or light chainor fragment thereof.

In some embodiments, at least one transgene encodes a small interferingnucleic acid. In some embodiments, the small interfering nucleic acid isa miRNA. In some embodiments, the small interfering nucleic acid is amiRNA sponge or TuD RNA that inhibits the activity of at least one miRNAin the subject or animal. In some embodiments, the miRNA is expressed ina cell of the target tissue. In some embodiments, the target tissue isskeletal muscle, heart, liver, pancreas, brain or lung. In someembodiments, the transgene expresses a transcript that comprises atleast one binding site for a miRNA, wherein the miRNA inhibits activityof the transgene, in a tissue other than the target tissue, byhybridizing to the binding site.

Reporter sequences that may be provided in a transgene include, withoutlimitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ),alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), luciferase, and others wellknown in the art. When associated with regulatory elements which drivetheir expression, the reporter sequences, provide signals detectable byconventional means, including enzymatic, radiographic, colorimetric,fluorescence or other spectrographic assays, fluorescent activating cellsorting assays and immunological assays, including enzyme linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) andimmunohistochemistry. For example, where the marker sequence is the LacZgene, the presence of the vector carrying the signal is detected byassays for β-galactosidase activity. Where the transgene is greenfluorescent protein or luciferase, the vector carrying the signal may bemeasured visually by color or light production in a luminometer. Suchreporters can, for example, be useful in verifying the tissue-specifictargeting capabilities and tissue specific promoter regulatory activityof an rAAV.

In some aspects, the disclosure provides rAAV vectors for use in methodsof preventing or treating one or more genetic deficiencies ordysfunctions in a mammal, such as for example, a polypeptide deficiencyor polypeptide excess in a mammal, and particularly for treating orreducing the severity or extent of deficiency in a human manifesting oneor more of the disorders linked to a deficiency in such polypeptides incells and tissues. The method involves administration of an rAAV vectorthat encodes one or more therapeutic peptides, polypeptides, siRNAs,microRNAs, antisense nucleotides, etc. in a pharmaceutically-acceptablecarrier to the subject in an amount and for a period of time sufficientto treat the deficiency or disorder in the subject suffering from such adisorder.

Thus, the disclosure embraces the delivery of rAAV vectors encoding oneor more peptides, polypeptides, or proteins, which are useful for thetreatment or prevention of disease states in a mammalian subject.Exemplary therapeutic proteins include one or more polypeptides selectedfrom the group consisting of growth factors, interleukins, interferons,anti-apoptosis factors, cytokines, anti-diabetic factors, anti-apoptosisagents, coagulation factors, anti-tumor factors. Other non-limitingexamples of therapeutic proteins include BDNF, CNTF, CSF, EGF, FGF,G-SCF, GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF,VEGF, TGF-B2, TNF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(187A), viral IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16 IL-17, and IL-18.

The rAAV vectors may comprise a gene to be transferred to a subject totreat a disease associated with reduced expression, lack of expressionor dysfunction of the gene. Exemplary genes and associated diseasestates include, but are not limited to: glucose-6-phosphatase,associated with glycogen storage deficiency type 1A;phosphoenolpyruvate-carboxykinase, associated with Pepck deficiency;galactose-1 phosphate uridyl transferase, associated with galactosemia;phenylalanine hydroxylase, associated with phenylketonuria; branchedchain alpha-ketoacid dehydrogenase, associated with Maple syrup urinedisease; fumarylacetoacetate hydrolase, associated with tyrosinemia type1; methylmalonyl-CoA mutase, associated with methylmalonic acidemia;medium chain acyl CoA dehydrogenase, associated with medium chain acetylCoA deficiency; omithine transcarbamylase, associated with omithinetranscarbamylase deficiency; argininosuccinic acid synthetase,associated with citrullinemia; low density lipoprotein receptor protein,associated with familial hypercholesterolemia;UDP-glucouronosyltransferase, associated with Crigler-Najjar disease;adenosine deaminase, associated with severe combined immunodeficiencydisease; hypoxanthine guanine phosphoribosyl transferase, associatedwith Gout and Lesch-Nyan syndrome; biotinidase, associated withbiotinidase deficiency; beta-galactosidase, associated with GM1gangliosidosis; beta-hexosaminidase A and B, associated with Tay-Sachsdisease and Sandhoff disease; beta-glucocerebrosidase, associated withGaucher disease; beta-glucuronidase, associated with Sly syndrome;peroxisome membrane protein 70 kDa, associated with Zellweger syndrome;porphobilinogen deaminase, associated with acute intermittent porphyria;alpha-1 antitrypsin for treatment of alpha-1 antitrypsin deficiency(emphysema); erythropoietin for treatment of anemia due to thalassemiaor to renal failure; vascular endothelial growth factor, angiopoietin-1,and fibroblast growth factor for the treatment of ischemic diseases;thrombomodulin and tissue factor pathway inhibitor for the treatment ofoccluded blood vessels as seen in, for example, atherosclerosis,thrombosis, or embolisms; aromatic amino acid decarboxylase (AADC), andtyrosine hydroxylase (TH) for the treatment of Parkinson's disease; thebeta adrenergic receptor, anti-sense to, or a mutant form of,phospholamban, the sarco(endo)plasmic reticulum adenosinetriphosphatase-2 (SERCA2), and the cardiac adenylyl cyclase for thetreatment of congestive heart failure; a tumor suppessor gene such asp53 for the treatment of various cancers; a cytokine such as one of thevarious interleukins for the treatment of inflammatory and immunedisorders and cancers; dystrophin or minidystrophin and utrophin orminiutrophin for the treatment of muscular dystrophies; and, insulin forthe treatment of diabetes.

In some embodiments, the rAAV vectors may comprise a gene encoding anantigen-binding protein, such as an immunoglobulin heavy chain or lightchain or fragment thereof, e.g., that may be used for therapeuticpurposes. In some embodiments, the protein is a single chain Fv fragmentor Fv-Fc fragment. Accordingly, in some embodiments, the rAAV can beused to infect cells are of target tissue (e.g., muscle tissue) toengineer cells of the tissue to express an antigen-binding protein, suchas an antibody or fragment thereof. In some embodiments, to generaterAAVs that express the antibodies or antigen binding fragments, cDNAsengineered to express such proteins will be sucloned into an appropriateplasmid backbone and packaged into an rAAV.

In some embodiments, the rAAV vectors may comprise a gene or genesencoding genome editing enzymes or related molecules. As used herein,“genome editing” refers to adding, disrupting or changing genomicsequences (e.g., a gene sequence). In some embodiments, genome editingis performed using engineered proteins and related molecules. In someaspects, genome editing comprises the use of engineered nucleases tocleave a target genomic locus. In some embodiments, genome editingfurther comprises inserting, deleting, mutating or substituting nucleicacid residues at a cleaved locus. In some embodiments, inserting,deleting, mutating or substituting nucleic acid residues at a cleavedlocus is accomplished through endogenous cellular mechanisms such ashomologous recombination (HR) and non-homologous end joining (NHEJ).Exemplary genome editing technologies include, but are not limited toTranscription Activator-like Effector Nucleases (TALENs), Zinc FingerNucleases (ZFNs), engineered meganuclease re-engineered homingendonucleases and the CRISPR/Cas system. In some embodiments, the rAAVmay comprise a gene or genes encoding proteins or molecules related toTALENs, including but not limited to transcription activator-likeeffectors (TALEs) and restriction endonucleases (e.g., FokI). In someembodiments, the rAAV may comprise a gene or genes encoding proteins ormolecules related to ZFNs, including but not limited to proteinscomprising the Cys₂His₂ fold group (for example Zif268 (EGR1)), andrestriction endonucleases (e.g., FokI). In some embodiments, the rAAVmay comprise a gene or genes encoding proteins or molecules related tothe CRISPR/Cas system, including but not limited to Cas9,Cas6, dCas9,CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).

The rAAVs of the disclosure can be used to restore the expression ofgenes that are reduced in expression, silenced, or otherwisedysfunctional in a subject (e.g., a tumor suppressor that has beensilenced in a subject having cancer). The rAAVs of the disclosure canalso be used to knockdown the expression of genes that are aberrantlyexpressed in a subject (e.g., an oncogene that is expressed in a subjecthaving cancer). In some embodiments, an rAAV vector comprising a nucleicacid encoding a gene product associated with cancer (e.g., tumorsuppressors) may be used to treat the cancer, by administering a rAAVharboring the rAAV vector to a subject having the cancer. In someembodiments, an rAAV vector comprising a nucleic acid encoding a smallinterfering nucleic acid (e.g., shRNAs, miRNAs) that inhibits theexpression of a gene product associated with cancer (e.g., oncogenes)may be used to treat the cancer, by administering a rAAV harboring therAAV vector to a subject having the cancer. In some embodiments, a rAAVvector comprising a nucleic acid encoding a gene product associated withcancer (or a functional RNA that inhibits the expression of a geneassociated with cancer) may be used for research purposes, e.g., tostudy the cancer or to identify therapeutics that treat the cancer. Thefollowing is a non-limiting list of exemplary genes known to beassociated with the development of cancer (e.g., oncogenes and tumorsuppressors): AARS, ABCB1, ABCC4, ABI2, ABL1, ABL2, ACK1, ACP2, ACY1,ADSL, AK1, AKR1C2, AKT1, ALB, ANPEP, ANXA5, ANXA7, AP2M1, APC, ARHGAP5,ARHGEF5, ARID4A, ASNS, ATF4, ATM, ATP5B, ATP5O, AXL, BARD1, BAX, BCL2,BHLHB2, BLMH, BRAF, BRCA1, BRCA2, BTK, CANX, CAP1, CAPN1, CAPNS1, CAV1,CBFB, CBLB, CCL2, CCND1, CCND2, CCND3, CCNE1, CCT5, CCYR61, CD24, CD44,CD59, CDC20, CDC25, CDC25A, CDC25B, CDC2L5, CDK10, CDK4, CDK5, CDK9,CDKL1, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2D, CEBPG, CENPC1,CGRRF1, CHAF1A, CIB1, CKMT1, CLK1, CLK2, CLK3, CLNS1A, CLTC, COL1A1,COL6A3, COX6C, COX7A2, CRAT, CRHR1, CSF1R, CSK, CSNK1G2, CTNNA1, CTNNB1,CTPS, CTSC, CTSD, CUL1, CYR61, DCC, DCN, DDX10, DEK, DHCR7, DHRS2, DHX8,DLG3, DVL1, DVL3, E2F1, E2F3, E2F5, EGFR, EGR1, EIF5, EPHA2, ERBB2,ERBB3, ERBB4, ERCC3, ETV1, ETV3, ETV6, F2R, FASTK, FBN1, FBN2, FES,FGFR1, FGR, FKBP8, FN1, FOS, FOSL1, FOSL2, FOXG1A, FOXO1A, FRAP1, FRZB,FTL, FZD2, FZD5, FZD9, G22P1, GAS6, GCN5L2, GDF15, GNA13, GNAS, GNB2,GNB2L1, GPR39, GRB2, GSK3A, GSPT1, GTF2I, HDAC1, HDGF, HMMR, HPRT1, HRB,HSPA4, HSPA5, HSPA8, HSPB1, HSPH1, HYAL1, HYOU1, ICAM1, ID1, ID2, IDUA,IER3, IFITM1, IGF1R, IGF2R, IGFBP3, IGFBP4, IGFBP5, IL1B, ILK, ING1,IRF3, ITGA3, ITGA6, ITGB4, JAK1, JARID1A, JUN, JUNB, JUND, K-ALPHA-1,KIT, KITLG, KLK10, KPNA2, KRAS2, KRT18, KRT2A, KRT9, LAMB1, LAMP2, LCK,LCN2, LEP, LITAF, LRPAP1, LTF, LYN, LZTR1, MADH1, MAP2K2, MAP3K8,MAPK12, MAPK13, MAPKAPK3, MAPRE1, MARS, MAS1, MCC, MCM2, MCM4, MDM2,MDM4, MET, MGST1, MICB, MLLT3, MME, MMP1, MMP14, MMP17, MMP2, MNDA,MSH2, MSH6, MT3, MYB, MYBL1, MYBL2, MYC, MYCL1, MYCN, MYD88, MYL9, MYLK,NEO1, NF1, NF2, NFKB1, NFKB2, NFSF7, NID, NINJ1, NMBR, NME1, NME2, NME3,NOTCH1, NOTCH2, NOTCH4, NPM1, NQO1, NR1D1, NR2F1, NR2F6, NRAS, NRG1,NSEP1, OSM, PA2G4, PABPC1, PCNA, PCTK1, PCTK2, PCTK3, PDGFA, PDGFB,PDGFRA, PDPK1, PEA15, PFDN4, PFDN5, PGAM1, PHB, PIK3CA, PIK3CB, PIK3CG,PIM1, PKM2, PKMYT1, PLK2, PPARD, PPARG, PPIH, PPP1CA, PPP2R5A, PRDX2,PRDX4, PRKAR1A, PRKCBP1, PRNP, PRSS15, PSMA1, PTCH, PTEN, PTGS1, PTMA,PTN, PTPRN, RAB5A, RAC1, RAD50, RAF1, RALBP1, RAP1A, RARA, RARB,RASGRF1, RB1, RBBP4, RBL2, REA, REL, RELA, RELB, RET, RFC2, RGS19, RHOA,RHOB, RHOC, RHOD, RIPK1, RPN2, RPS6KB1, RRM1, SARS, SELENBP1, SEMA3C,SEMA4D, SEPP1, SERPINH1, SFN, SFPQ, SFRS7, SHB, SHH, SIAH2, SIVA, SIVATP53, SKI, SKIL, SLC16A1, SLC1A4, SLC20A1, SMO, SMPD1, SNAI2, SND1,SNRPB2, SOCS1, SOCS3, SOD1, SORT1, SPINT2, SPRY2, SRC, SRPX, STAT1,STAT2, STAT3, STAT5B, STC1, TAF1, TBL3, TBRG4, TCF1, TCF7L2, TFAP2C,TFDP1, TFDP2, TGFA, TGFB1, TGFBI, TGFBR2, TGFBR3, THBS1, TIE, TIMP1,TIMP3, TJP1, TK1, TLE1, TNF, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF1B,TNFRSF6, TNFSF7, TNK1, TOB1, TP53, TP53BP2, TP53I3, TP73, TPBG, TPT1,TRADD, TRAM1, TRRAP, TSG101, TUFM, TXNRD1, TYRO3, UBC, UBE2L6, UCHL1,USP7, VDAC1, VEGF, VHL, VIL2, WEE1, WNT1, WNT2, WNT2B, WNT3, WNT5A, WT1,XRCC1, YES1, YWHAB, YWHAZ, ZAP70, and ZNF9.

A rAAV vector may comprise as a transgene, a nucleic acid encoding aprotein or functional RNA that modulates apoptosis. The following is anon-limiting list of genes associated with apoptosis and nucleic acidsencoding the products of these genes and their homologues and encodingsmall interfering nucleic acids (e.g., shRNAs, miRNAs) that inhibit theexpression of these genes and their homologues are useful as transgenesin certain embodiments of the disclosure: RPS27A, ABL1, AKT1, APAF1,BAD, BAG1, BAG3, BAG4, BAK1, BAX, BCL10, BCL2, BCL2A1, BCL2L1, BCL2L10,BCL2L11, BCL2L12, BCL2L13, BCL2L2, BCLAF1, BFAR, BID, BIK, NAIP, BIRC2,BIRC3, XIAP, BIRC5, BIRC6, BIRC7, BIRC8, BNIP1, BNIP2, BNIP3, BNIP3L,BOK, BRAF, CARD10, CARD11, NLRC4, CARD14, NOD2, NOD1, CARD6, CARDS,CARD9, CASP1, CASP10, CASP14, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7,CASP8, CASP9, CFLAR, CIDEA, CIDEB, CRADD, DAPK1, DAPK2, DFFA, DFFB,FADD, GADD45A, GDNF, HRK, IGF1R, LTA, LTBR, MCL1, NOL3, PYCARD, RIPK1,RIPK2, TNF, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11B,TNFRSF12A, TNFRSF14, TNFRSF19, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF25,CD40, FAS, TNFRSF6B, CD27, TNFRSF9, TNFSF10, TNFSF14, TNFSF18, CD4OLG,FASLG, CD70, TNFSF8, TNFSF9, TP53, TP53BP2, TP73, TP63, TRADD, TRAF1,TRAF2, TRAF3, TRAF4, and TRAF5.

In some aspects, the disclosure relates to methods and compositions fortreating CNS-related disorders. As used herein, a “CNS-related disorder”is a disease or condition of the central nervous system. A CNS-relateddisorder may affect the spinal cord (e.g., a myelopathy), brain (e.g., aencephalopathy) or tissues surrounding the brain and spinal cord. ACNS-related disorder may be of a genetic origin, either inherited oracquired through a somatic mutation. A CNS-related disorder may be apsychological condition or disorder, e.g., Attention DeficientHyperactivity Disorder, Autism Spectrum Disorder, Mood Disorder,Schizophrenia, Depression, Rett Syndrome, etc. A CNS-related disordermay be an autoimmune disorder. A CNS-related disorder may also be acancer of the CNS, e.g., brain cancer. A CNS-related disorder that is acancer may be a primary cancer of the CNS, e.g., an astrocytoma,glioblastomas, etc., or may be a cancer that has metastasized to CNStissue, e.g., a lung cancer that has metastasized to the brain. Furthernon-limiting examples of CNS-related disorders, include Huntington'sdisease, Parkinson's Disease, Lysosomal Storage Disease, Ischemia,Neuropathic Pain, Amyotrophic lateral sclerosis (ALS), MultipleSclerosis (MS), and Canavan disease (CD).

In some embodiments, the disclosure relates to a rAAV vector comprisinga transgene, a nucleic acid encoding a protein or functional RNA usefulfor the treatment of a condition, disease or disorder associated withthe central nervous system (CNS). The following is a non-limiting listof genes associated with CNS disease: DRD2, GRIA1, GRIA2,GRIN1, SLC1A1,SYP, SYT1, CHRNA7, 3Rtau/4rTUS, APP, BAX, BCL-2, GRIK1, GFAP, IL-1,AGER, associated with Alzheimer's Disease; UCH-L1, SKP1, EGLN1, Nurr-1,BDNF, TrkB,gstml, S10613, associated with Parkinson's Disease;huntingtin (Htt), IT15, PRNP, JPH3, TBP, ATXN1, ATXN2, ATXN3, Atrophin1, FTL, TITF-1, associated with Huntington's Disease; FXN, associatedwith Freidrich's ataxia; ASPA, associated with Canavan's Disease; DMD,associated with muscular dystrophy; and SMN1, UBE1, DYNC1H1 associatedwith spinal muscular atrophy.

The skilled artisan will also realize that in the case of transgenesencoding proteins or polypeptides, that mutations that results inconservative amino acid substitutions may be made in a transgene toprovide functionally equivalent variants, or homologs of a protein orpolypeptide. In some aspects the disclosure embraces sequencealterations that result in conservative amino acid substitution of atransgene. In some embodiments, the transgene comprises a gene having adominant negative mutation. For example, a transgene may express amutant protein that interacts with the same elements as a wild-typeprotein, and thereby blocks some aspect of the function of the wild-typeprotein.

Useful transgene products also include miRNAs. miRNAs and other smallinterfering nucleic acids regulate gene expression via target RNAtranscript cleavage/degradation or translational repression of thetarget messenger RNA (mRNA). miRNAs are natively expressed, typically asfinal 19-25 non-translated RNA products. miRNAs exhibit their activitythrough sequence-specific interactions with the 3′ untranslated regions(UTR) of target mRNAs. These endogenously expressed miRNAs form hairpinprecursors which are subsequently processed into a miRNA duplex, andfurther into a “mature” single stranded miRNA molecule. This maturemiRNA guides a multiprotein complex, miRISC, which identifies targetsite, e.g., in the 3′ UTR regions, of target mRNAs based upon theircomplementarity to the mature miRNA.

The following non-limiting list of miRNA genes, and their homologues,are useful as transgenes or as targets for small interfering nucleicacids encoded by transgenes (e.g., miRNA sponges, antisenseoligonucleotides, TuD RNAs) in certain embodiments of the methods:hsa-let-7a, hsa-let-7a*, hsa-let-7b, hsa-let-7b*, hsa-let-7c,hsa-let-7c*, hsa-let-7d, hsa-let-7d*, hsa-let-7e, hsa-let-7e*,hsa-let-7f, hsa-let-7f-1*, hsa-let-7f-2*, hsa-let-7g, hsa-let-7g*,hsa-let-7i, hsa-let-7i*, hsa-miR-1, hsa-miR-100, hsa-miR-100*,hsa-miR-101, hsa-miR-101*, hsa-miR-103, hsa-miR-105, hsa-miR-105*,hsa-miR-106a, hsa-miR-106a*, hsa-miR-106b, hsa-miR-106b*, hsa-miR-107,hsa-miR-10a, hsa-miR-10a*, hsa-miR-10b, hsa-miR-10b*, hsa-miR-1178,hsa-miR-1179, hsa-miR-1180, hsa-miR-1181, hsa-miR-1182, hsa-miR-1183,hsa-miR-1184, hsa-miR-1185, hsa-miR-1197, hsa-miR-1200, hsa-miR-1201,hsa-miR-1202, hsa-miR-1203, hsa-miR-1204, hsa-miR-1205, hsa-miR-1206,hsa-miR-1207-3p, hsa-miR-1207-5p, hsa-miR-1208, hsa-miR-122,hsa-miR-122*, hsa-miR-1224-3p, hsa-miR-1224-5p, hsa-miR-1225-3p,hsa-miR-1225-5p, hsa-miR-1226, hsa-miR-1226*, hsa-miR-1227,hsa-miR-1228, hsa-miR-1228*, hsa-miR-1229, hsa-miR-1231, hsa-miR-1233,hsa-miR-1234, hsa-miR-1236, hsa-miR-1237, hsa-miR-1238, hsa-miR-124,hsa-miR-124*, hsa-miR-1243, hsa-miR-1244, hsa-miR-1245, hsa-miR-1246,hsa-miR-1247, hsa-miR-1248, hsa-miR-1249, hsa-miR-1250, hsa-miR-1251,hsa-miR-1252, hsa-miR-1253, hsa-miR-1254, hsa-miR-1255a, hsa-miR-1255b,hsa-miR-1256, hsa-miR-1257, hsa-miR-1258, hsa-miR-1259, hsa-miR-125a-3p,hsa-miR-125a-5p, hsa-miR-125b, hsa-miR-125b-1*, hsa-miR-125b-2*,hsa-miR-126, hsa-miR-126*, hsa-miR-1260, hsa-miR-1261, hsa-miR-1262,hsa-miR-1263, hsa-miR-1264, hsa-miR-1265, hsa-miR-1266, hsa-miR-1267,hsa-miR-1268, hsa-miR-1269, hsa-miR-1270, hsa-miR-1271, hsa-miR-1272,hsa-miR-1273, hsa-miR-127-3p, hsa-miR-1274a, hsa-miR-1274b,hsa-miR-1275, hsa-miR-127-5p, hsa-miR-1276, hsa-miR-1277, hsa-miR-1278,hsa-miR-1279, hsa-miR-128, hsa-miR-1280, hsa-miR-1281, hsa-miR-1282,hsa-miR-1283, hsa-miR-1284, hsa-miR-1285, hsa-miR-1286, hsa-miR-1287,hsa-miR-1288, hsa-miR-1289, hsa-miR-129*, hsa-miR-1290, hsa-miR-1291,hsa-miR-1292, hsa-miR-1293, hsa-miR-129-3p, hsa-miR-1294, hsa-miR-1295,hsa-miR-129-5p, hsa-miR-1296, hsa-miR-1297, hsa-miR-1298, hsa-miR-1299,hsa-miR-1300, hsa-miR-1301, hsa-miR-1302, hsa-miR-1303, hsa-miR-1304,hsa-miR-1305, hsa-miR-1306, hsa-miR-1307, hsa-miR-1308, hsa-miR-130a,hsa-miR-130a*, hsa-miR-130b, hsa-miR-130b*, hsa-miR-132, hsa-miR-132*,hsa-miR-1321, hsa-miR-1322, hsa-miR-1323, hsa-miR-1324, hsa-miR-133a,hsa-miR-133b, hsa-miR-134, hsa-miR-135a, hsa-miR-135a*, hsa-miR-135b,hsa-miR-135b*, hsa-miR-136, hsa-miR-136*, hsa-miR-137, hsa-miR-138,hsa-miR-138-1*, hsa-miR-138-2*, hsa-miR-139-3p, hsa-miR-139-5p,hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR-141, hsa-miR-141*,hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-143, hsa-miR-143*, hsa-miR-144,hsa-miR-144*, hsa-miR-145, hsa-miR-145*, hsa-miR-146a, hsa-miR-146a*,hsa-miR-146b-3p, hsa-miR-146b-5p, hsa-miR-147, hsa-miR-147b,hsa-miR-148a, hsa-miR-148a*, hsa-miR-148b, hsa-miR-148b*, hsa-miR-149,hsa-miR-149*, hsa-miR-150, hsa-miR-150*, hsa-miR-151-3p, hsa-miR-151-5p,hsa-miR-152, hsa-miR-153, hsa-miR-154, hsa-miR-154*, hsa-miR-155,hsa-miR-155*, hsa-miR-15a, hsa-miR-15a*, hsa-miR-15b, hsa-miR-15b*,hsa-miR-16, hsa-miR-16-1*, hsa-miR-16-2*, hsa-miR-17, hsa-miR-17*,hsa-miR-181a, hsa-miR-181a*, hsa-miR-181a-2*, hsa-miR-181b,hsa-miR-181c, hsa-miR-181c*, hsa-miR-181d, hsa-miR-182, hsa-miR-182*,hsa-miR-1825, hsa-miR-1826, hsa-miR-1827, hsa-miR-183, hsa-miR-183*,hsa-miR-184, hsa-miR-185, hsa-miR-185*, hsa-miR-186, hsa-miR-186*,hsa-miR-187, hsa-miR-187*, hsa-miR-188-3p, hsa-miR-188-5p, hsa-miR-18a,hsa-miR-18a*, hsa-miR-18b, hsa-miR-18b*, hsa-miR-190, hsa-miR-190b,hsa-miR-191, hsa-miR-191*, hsa-miR-192, hsa-miR-192*, hsa-miR-193a-3p,hsa-miR-193a-5p, hsa-miR-193b, hsa-miR-193b*, hsa-miR-194, hsa-miR-194*,hsa-miR-195, hsa-miR-195*, hsa-miR-196a, hsa-miR-196a*, hsa-miR-196b,hsa-miR-197, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-199a-5p,hsa-miR-199b-5p, hsa-miR-19a, hsa-miR-19a*, hsa-miR-19b, hsa-miR-19b-1*,hsa-miR-19b-2*, hsa-miR-200a, hsa-miR-200a*, hsa-miR-200b,hsa-miR-200b*, hsa-miR-200c, hsa-miR-200c*, hsa-miR-202, hsa-miR-202*,hsa-miR-203, hsa-miR-204, hsa-miR-205, hsa-miR-206, hsa-miR-208a,hsa-miR-208b, hsa-miR-20a, hsa-miR-20a*, hsa-miR-20b, hsa-miR-20b*,hsa-miR-21, hsa-miR-21*, hsa-miR-210, hsa-miR-211, hsa-miR-212,hsa-miR-214, hsa-miR-214*, hsa-miR-215, hsa-miR-216a, hsa-miR-216b,hsa-miR-217, hsa-miR-218, hsa-miR-218-1*, hsa-miR-218-2*,hsa-miR-219-1-3p, hsa-miR-219-2-3p, hsa-miR-219-5p, hsa-miR-22,hsa-miR-22*, hsa-miR-220a, hsa-miR-220b, hsa-miR-220c, hsa-miR-221,hsa-miR-221*, hsa-miR-222, hsa-miR-222*, hsa-miR-223, hsa-miR-223*,hsa-miR-224, hsa-miR-23a, hsa-miR-23a*, hsa-miR-23b, hsa-miR-23b*,hsa-miR-24, hsa-miR-24-1*, hsa-miR-24-2*, hsa-miR-25, hsa-miR-25*,hsa-miR-26a, hsa-miR-26a-1*, hsa-miR-26a-2*, hsa-miR-26b, hsa-miR-26b*,hsa-miR-27a, hsa-miR-27a*, hsa-miR-27b, hsa-miR-27b*, hsa-miR-28-3p,hsa-miR-28-5p, hsa-miR-296-3p, hsa-miR-296-5p, hsa-miR-297, hsa-miR-298,hsa-miR-299-3p, hsa-miR-299-5p, hsa-miR-29a, hsa-miR-29a*, hsa-miR-29b,hsa-miR-29b-1*, hsa-miR-29b-2*, hsa-miR-29c, hsa-miR-29c*, hsa-miR-300,hsa-miR-301a, hsa-miR-301b, hsa-miR-302a, hsa-miR-302a*, hsa-miR-302b,hsa-miR-302b*, hsa-miR-302c, hsa-miR-302c*, hsa-miR-302d, hsa-miR-302d*,hsa-miR-302e, hsa-miR-302f, hsa-miR-30a, hsa-miR-30a*, hsa-miR-30b,hsa-miR-30b*, hsa-miR-30c, hsa-miR-30c-1*, hsa-miR-30c-2*, hsa-miR-30d,hsa-miR-30d*, hsa-miR-30e, hsa-miR-30e*, hsa-miR-31, hsa-miR-31*,hsa-miR-32, hsa-miR-32*, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c,hsa-miR-320d, hsa-miR-323-3p, hsa-miR-323-5p, hsa-miR-324-3p,hsa-miR-324-5p, hsa-miR-325, hsa-miR-326, hsa-miR-328, hsa-miR-329,hsa-miR-330-3p, hsa-miR-330-5p, hsa-miR-331-3p, hsa-miR-331-5p,hsa-miR-335, hsa-miR-335*, hsa-miR-337-3p, hsa-miR-337-5p,hsa-miR-338-3p, hsa-miR-338-5p, hsa-miR-339-3p, hsa-miR-339-5p,hsa-miR-33a, hsa-miR-33a*, hsa-miR-33b, hsa-miR-33b*, hsa-miR-340,hsa-miR-340*, hsa-miR-342-3p, hsa-miR-342-5p, hsa-miR-345, hsa-miR-346,hsa-miR-34a, hsa-miR-34a*, hsa-miR-34b, hsa-miR-34b*, hsa-miR-34c-3p,hsa-miR-34c-5p, hsa-miR-361-3p, hsa-miR-361-5p, hsa-miR-362-3p,hsa-miR-362-5p, hsa-miR-363, hsa-miR-363*, hsa-miR-365, hsa-miR-367,hsa-miR-367*, hsa-miR-369-3p, hsa-miR-369-5p, hsa-miR-370,hsa-miR-371-3p, hsa-miR-371-5p, hsa-miR-372, hsa-miR-373, hsa-miR-373*,hsa-miR-374a, hsa-miR-374a*, hsa-miR-374b, hsa-miR-374b*, hsa-miR-375,hsa-miR-376a, hsa-miR-376a*, hsa-miR-376b, hsa-miR-376c, hsa-miR-377,hsa-miR-377*, hsa-miR-378, hsa-miR-378*, hsa-miR-379, hsa-miR-379*,hsa-miR-380, hsa-miR-380*, hsa-miR-381, hsa-miR-382, hsa-miR-383,hsa-miR-384, hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR-410, hsa-miR-411,hsa-miR-411*, hsa-miR-412, hsa-miR-421, hsa-miR-422a, hsa-miR-423-3p,hsa-miR-423-5p, hsa-miR-424, hsa-miR-424*, hsa-miR-425, hsa-miR-425*,hsa-miR-429, hsa-miR-431, hsa-miR-431*, hsa-miR-432, hsa-miR-432*,hsa-miR-433, hsa-miR-448, hsa-miR-449a, hsa-miR-449b, hsa-miR-450a,hsa-miR-450b-3p, hsa-miR-450b-5p, hsa-miR-451, hsa-miR-452,hsa-miR-452*, hsa-miR-453, hsa-miR-454, hsa-miR-454*, hsa-miR-455-3p,hsa-miR-455-5p, hsa-miR-483-3p, hsa-miR-483-5p, hsa-miR-484,hsa-miR-485-3p, hsa-miR-485-5p, hsa-miR-486-3p, hsa-miR-486-5p,hsa-miR-487a, hsa-miR-487b, hsa-miR-488, hsa-miR-488*, hsa-miR-489,hsa-miR-490-3p, hsa-miR-490-5p, hsa-miR-491-3p, hsa-miR-491-5p,hsa-miR-492, hsa-miR-493, hsa-miR-493*, hsa-miR-494, hsa-miR-495,hsa-miR-496, hsa-miR-497, hsa-miR-497*, hsa-miR-498, hsa-miR-499-3p,hsa-miR-499-5p, hsa-miR-500, hsa-miR-500*, hsa-miR-501-3p,hsa-miR-501-5p, hsa-miR-502-3p, hsa-miR-502-5p, hsa-miR-503,hsa-miR-504, hsa-miR-505, hsa-miR-505*, hsa-miR-506, hsa-miR-507,hsa-miR-508-3p, hsa-miR-508-5p, hsa-miR-509-3-5p, hsa-miR-509-3p,hsa-miR-509-5p, hsa-miR-510, hsa-miR-511, hsa-miR-512-3p,hsa-miR-512-5p, hsa-miR-513a-3p, hsa-miR-513a-5p, hsa-miR-513b,hsa-miR-513c, hsa-miR-514, hsa-miR-515-3p, hsa-miR-515-5p,hsa-miR-516a-3p, hsa-miR-516a-5p, hsa-miR-516b, hsa-miR-517*,hsa-miR-517a, hsa-miR-517b, hsa-miR-517c, hsa-miR-518a-3p,hsa-miR-518a-5p, hsa-miR-518b, hsa-miR-518c, hsa-miR-518c*,hsa-miR-518d-3p, hsa-miR-518d-5p, hsa-miR-518e, hsa-miR-518e*,hsa-miR-518f, hsa-miR-518f*, hsa-miR-519a, hsa-miR-519b-3p,hsa-miR-519c-3p, hsa-miR-519d, hsa-miR-519e, hsa-miR-519e*,hsa-miR-520a-3p, hsa-miR-520a-5p, hsa-miR-520b, hsa-miR-520c-3p,hsa-miR-520d-3p, hsa-miR-520d-5p, hsa-miR-520e, hsa-miR-520f,hsa-miR-520g, hsa-miR-520h, hsa-miR-521, hsa-miR-522, hsa-miR-523,hsa-miR-524-3p, hsa-miR-524-5p, hsa-miR-525-3p, hsa-miR-525-5p,hsa-miR-526b, hsa-miR-526b*, hsa-miR-532-3p, hsa-miR-532-5p,hsa-miR-539, hsa-miR-541, hsa-miR-541*, hsa-miR-542-3p, hsa-miR-542-5p,hsa-miR-543, hsa-miR-544, hsa-miR-545, hsa-miR-545*, hsa-miR-548a-3p,hsa-miR-548a-5p, hsa-miR-548b-3p, hsa-miR-548b-5p, hsa-miR-548c-3p,hsa-miR-548c-5p, hsa-miR-548d-3p, hsa-miR-548d-5p, hsa-miR-548e,hsa-miR-548f, hsa-miR-548g, hsa-miR-548h, hsa-miR-5481, hsa-miR-548j,hsa-miR-548k, hsa-miR-5481, hsa-miR-548m, hsa-miR-548n, hsa-miR-548o,hsa-miR-548p, hsa-miR-549, hsa-miR-550, hsa-miR-550*, hsa-miR-551a,hsa-miR-551b, hsa-miR-551b*, hsa-miR-552, hsa-miR-553, hsa-miR-554,hsa-miR-555, hsa-miR-556-3p, hsa-miR-556-5p, hsa-miR-557, hsa-miR-558,hsa-miR-559, hsa-miR-561, hsa-miR-562, hsa-miR-563, hsa-miR-564,hsa-miR-566, hsa-miR-567, hsa-miR-568, hsa-miR-569, hsa-miR-570,hsa-miR-571, hsa-miR-572, hsa-miR-573, hsa-miR-574-3p, hsa-miR-574-5p,hsa-miR-575, hsa-miR-576-3p, hsa-miR-576-5p, hsa-miR-577, hsa-miR-578,hsa-miR-579, hsa-miR-580, hsa-miR-581, hsa-miR-582-3p, hsa-miR-582-5p,hsa-miR-583, hsa-miR-584, hsa-miR-585, hsa-miR-586, hsa-miR-587,hsa-miR-588, hsa-miR-589, hsa-miR-589*, hsa-miR-590-3p, hsa-miR-590-5p,hsa-miR-591, hsa-miR-592, hsa-miR-593, hsa-miR-593*, hsa-miR-595,hsa-miR-596, hsa-miR-597, hsa-miR-598, hsa-miR-599, hsa-miR-600,hsa-miR-601, hsa-miR-602, hsa-miR-603, hsa-miR-604, hsa-miR-605,hsa-miR-606, hsa-miR-607, hsa-miR-608, hsa-miR-609, hsa-miR-610,hsa-miR-611, hsa-miR-612, hsa-miR-613, hsa-miR-614, hsa-miR-615-3p,hsa-miR-615-5p, hsa-miR-616, hsa-miR-616*, hsa-miR-617, hsa-miR-618,hsa-miR-619, hsa-miR-620, hsa-miR-621, hsa-miR-622, hsa-miR-623,hsa-miR-624, hsa-miR-624*, hsa-miR-625, hsa-miR-625*, hsa-miR-626,hsa-miR-627, hsa-miR-628-3p, hsa-miR-628-5p, hsa-miR-629, hsa-miR-629*,hsa-miR-630, hsa-miR-631, hsa-miR-632, hsa-miR-633, hsa-miR-634,hsa-miR-635, hsa-miR-636, hsa-miR-637, hsa-miR-638, hsa-miR-639,hsa-miR-640, hsa-miR-641, hsa-miR-642, hsa-miR-643, hsa-miR-644,hsa-miR-645, hsa-miR-646, hsa-miR-647, hsa-miR-648, hsa-miR-649,hsa-miR-650, hsa-miR-651, hsa-miR-652, hsa-miR-653, hsa-miR-654-3p,hsa-miR-654-5p, hsa-miR-655, hsa-miR-656, hsa-miR-657, hsa-miR-658,hsa-miR-659, hsa-miR-660, hsa-miR-661, hsa-miR-662, hsa-miR-663,hsa-miR-663b, hsa-miR-664, hsa-miR-664*, hsa-miR-665, hsa-miR-668,hsa-miR-671-3p, hsa-miR-671-5p, hsa-miR-675, hsa-miR-7, hsa-miR-708,hsa-miR-708*, hsa-miR-7-1*, hsa-miR-7-2*, hsa-miR-720, hsa-miR-744,hsa-miR-744*, hsa-miR-758, hsa-miR-760, hsa-miR-765, hsa-miR-766,hsa-miR-767-3p, hsa-miR-767-5p, hsa-miR-768-3p, hsa-miR-768-5p,hsa-miR-769-3p, hsa-miR-769-5p, hsa-miR-770-5p, hsa-miR-802,hsa-miR-873, hsa-miR-874, hsa-miR-875-3p, hsa-miR-875-5p,hsa-miR-876-3p, hsa-miR-876-5p, hsa-miR-877, hsa-miR-877*,hsa-miR-885-3p, hsa-miR-885-5p, hsa-miR-886-3p, hsa-miR-886-5p,hsa-miR-887, hsa-miR-888, hsa-miR-888*, hsa-miR-889, hsa-miR-890,hsa-miR-891 a, hsa-miR-891b, hsa-miR-892a, hsa-miR-892b, hsa-miR-9,hsa-miR-9*, hsa-miR-920, hsa-miR-921, hsa-miR-922, hsa-miR-923,hsa-miR-924, hsa-miR-92a, hsa-miR-92a-1*, hsa-miR-92a-2*, hsa-miR-92b,hsa-miR-92b*, hsa-miR-93, hsa-miR-93*, hsa-miR-933, hsa-miR-934,hsa-miR-935, hsa-miR-936, hsa-miR-937, hsa-miR-938, hsa-miR-939,hsa-miR-940, hsa-miR-941, hsa-miR-942, hsa-miR-943, hsa-miR-944,hsa-miR-95, hsa-miR-96, hsa-miR-96*, hsa-miR-98, hsa-miR-99a,hsa-miR-99a*, hsa-miR-99b, and hsa-miR-99b*.

A miRNA inhibits the function of the mRNAs it targets and, as a result,inhibits expression of the polypeptides encoded by the mRNAs. Thus,blocking (partially or totally) the activity of the miRNA (e.g.,silencing the miRNA) can effectively induce, or restore, expression of apolypeptide whose expression is inhibited (derepress the polypeptide).In one embodiment, derepression of polypeptides encoded by mRNA targetsof a miRNA is accomplished by inhibiting the miRNA activity in cellsthrough any one of a variety of methods. For example, blocking theactivity of a miRNA can be accomplished by hybridization with a smallinterfering nucleic acid (e.g., antisense oligonucleotide, miRNA sponge,TuD RNA) that is complementary, or substantially complementary to, themiRNA, thereby blocking interaction of the miRNA with its target mRNA.As used herein, an small interfering nucleic acid that is substantiallycomplementary to a miRNA is one that is capable of hybridizing with amiRNA, and blocking the miRNA's activity. In some embodiments, an smallinterfering nucleic acid that is substantially complementary to a miRNAis an small interfering nucleic acid that is complementary with themiRNA at all but 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, or 18 bases. In some embodiments, an small interfering nucleic acidsequence that is substantially complementary to a miRNA, is an smallinterfering nucleic acid sequence that is complementary with the miRNAat, at least, one base.

A “miRNA Inhibitor” is an agent that blocks miRNA function, expressionand/or processing. For instance, these molecules include but are notlimited to microRNA specific antisense, microRNA sponges, tough decoyRNAs (TuD RNAs) and microRNA oligonucleotides (double-stranded, hairpin,short oligonucleotides) that inhibit miRNA interaction with a Droshacomplex. MicroRNA inhibitors can be expressed in cells from a transgenesof a rAAV vector, as discussed above. MicroRNA sponges specificallyinhibit miRNAs through a complementary heptameric seed sequence (Ebert,M.S. Nature Methods, Epub Aug., 12, 2007;). In some embodiments, anentire family of miRNAs can be silenced using a single sponge sequence.TuD RNAs achieve efficient and long-term-suppression of specific miRNAsin mammalian cells (See, e.g., Takeshi Haraguchi, et al., Nucleic AcidsResearch, 2009, Vol. 37, No. 6 e43, the contents of which relating toTuD RNAs are incorporated herein by reference). Other methods forsilencing miRNA function (derepression of miRNA targets) in cells willbe apparent to one of ordinary skill in the art.

In some embodiments, the cloning capacity of the recombinant RNA vectormay limited and a desired coding sequence may require the completereplacement of the virus's 4.8 kilobase genome. Large genes may,therefore, not be suitable for use in a standard recombinant AAV vector,in some cases. The skilled artisan will appreciate that options areavailable in the art for overcoming a limited coding capacity. Forexample, the AAV ITRs of two genomes can anneal to form head to tailconcatamers, almost doubling the capacity of the vector. Insertion ofsplice sites allows for the removal of the ITRs from the transcript.Other options for overcoming a limited cloning capacity will be apparentto the skilled artisan.

Somatic Transgenic Animal Models Produced Using rAAV-Based Gene Transfer

The disclosure also involves the production of somatic transgenic animalmodels of disease using recombinant Adeno-Associated Virus (rAAV) basedmethods. The methods are based, at least in part, on the observationthat AAV serotypes and variants thereof mediate efficient and stablegene transfer in a tissue specific manner in adult animals. The rAAVelements (capsid, promoter, transgene products) are combined to achievesomatic transgenic animal models that express a stable transgene in atime and tissue specific manner. The somatic transgenic animal producedby the methods of the disclosure can serve as useful models of humandisease, pathological state, and/or to characterize the effects of genefor which the function (e.g., tissue specific, disease role) is unknownor not fully understood. For example, an animal (e.g., mouse) can beinfected at a distinct developmental stage (e.g., age) with a rAAVcomprising a capsid having a specific tissue targeting capability (e.g.,liver, heart, pancreas) and a transgene having a tissue specificpromoter driving expression of a gene involved in disease. Uponinfection, the rAAV infects distinct cells of the target tissue andproduces the product of the transgene.

In some embodiments, the sequence of the coding region of a transgene ismodified. The modification may alter the function of the product encodedby the transgene. The effect of the modification can then be studied invivo by generating a somatic transgenic animal model using the methodsdisclosed herein. In some embodiments, modification of the sequence ofcoding region is a nonsense mutation that results in a fragment (e.g., atruncated version). In other cases, the modification is a missensemutation that results in an amino acid substitution. Other modificationsare possible and will be apparent to the skilled artisan.

In some embodiments, the transgene causes a pathological state. Atransgene that causes a pathological state is a gene whose product has arole in a disease or disorder (e.g., causes the disease or disorder,makes the animal susceptible to the disease or disorder) and/or mayinduce the disease or disorder in the animal. The animal can then beobserved to evaluate any number of aspects of the disease (e.g.,progression, response to treatment, etc.). These examples are not meantto be limiting, other aspects and examples are disclosed herein anddescribed in more detail below.

The disclosure in some aspects, provide methods for producing somatictransgenic animal models through the targeted destruction of specificcell types. For example, models of type 1 diabetes can be produced bythe targeted destruction of pancreatic Beta-islets. In other examples,the targeted destruction of specific cell types can be used to evaluatethe role of specific cell types on human disease. In this regard,transgenes that encode cellular toxins (e.g., diphtheria toxin A (DTA))or pro-apoptotic genes (NTR, Box, etc.) can be useful as transgenes forfunctional ablation of specific cell types. Other exemplary transgenes,whose products kill cells are embraced by the methods disclosed hereinand will be apparent to one of ordinary skill in the art.

The disclosure in some aspects, provides methods for producing somatictransgenic animal models to study the long-term effects ofover-expression or knockdown of genes. The long term over expression orknockdown (e.g., by shRNA, miRNA, miRNA inhibitor, etc.) of genes inspecific target tissues can disturb normal metabolic balance andestablish a pathological state, thereby producing an animal model of adisease, such as, for example, cancer. The disclosure in some aspects,provides methods for producing somatic transgenic animal models to studythe long-term effects of over-expression or knockdown of gene ofpotential oncogenes and other genes to study tumorigenesis and genefunction in the targeted tissues. Useful transgene products includeproteins that are known to be associated with cancer and smallinterfering nucleic acids inhibiting the expression of such proteins.Other suitable transgenes may be readily selected by one of skill in theart provided that they are useful for creating animal models oftissue-specific pathological state and/or disease.

Recombinant AAV Administration Methods

The rAAVs may be delivered to a subject in compositions according to anyappropriate methods known in the art. The rAAV, preferably suspended ina physiologically compatible carrier (i.e., in a composition), may beadministered to a subject, i.e. host animal, such as a human, mouse,rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig,hamster, chicken, turkey, or a non-human primate (e.g., Macaque). Insome embodiments a host animal does not include a human.

Delivery of the rAAVs to a mammalian subject may be by, for example,intramuscular injection or by administration into the bloodstream of themammalian subject. Administration into the bloodstream may be byinjection into a vein, an artery, or any other vascular conduit. In someembodiments, the rAAVs are administered into the bloodstream by way ofisolated limb perfusion, a technique well known in the surgical arts,the method essentially enabling the artisan to isolate a limb from thesystemic circulation prior to administration of the rAAV virions. Avariant of the isolated limb perfusion technique, described in U.S. Pat.No. 6,177,403, can also be employed by the skilled artisan to administerthe virions into the vasculature of an isolated limb to potentiallyenhance transduction into muscle cells or tissue. Moreover, in certaininstances, it may be desirable to deliver the virions to the CNS of asubject. By “CNS” is meant all cells and tissue of the brain and spinalcord of a vertebrate. Thus, the term includes, but is not limited to,neuronal cells, glial cells, astrocytes, cerebrospinal fluid (CSF),interstitial spaces, bone, cartilage and the like. Recombinant AAVs maybe delivered directly to the CNS or brain by injection into, e.g., theventricular region, as well as to the striatum (e.g., the caudatenucleus or putamen of the striatum), spinal cord and neuromuscularjunction, or cerebellar lobule, with a needle, catheter or relateddevice, using neurosurgical techniques known in the art, such as bystereotactic injection (see, e.g., Stein et al., J Virol 73:3424-3429,1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat.Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther.11:2315-2329, 2000).

Aspects of the disclosure relate to compositions comprising arecombinant AAV comprising a heterologous targeting peptide. In someembodiments, the heterologous targeting peptide is N-terminally graftedonto a capsid protein. In some embodiments, the an N-terminally graftedheterologous targeting peptide is present on all three capsid proteins(e.g., VP1, VP2, VP3) of the rAAV. In some embodiments, the N-terminallygrafted heterologous targeting peptide is present on two of the capsidproteins (e.g., VP2 and VP3) of the rAAV. In some embodiments, theN-terminally grafted heterologous targeting peptide is present on asingle capsid protein of the rAAV. In some embodiments, the N-terminallygrafted heterologous targeting peptide is present on the VP2 capsidprotein of the rAAV. In some embodiments, the composition furthercomprises a pharmaceutically acceptable carrier.

The compositions of the disclosure may comprise an rAAV alone, or incombination with one or more other viruses (e.g., a second rAAV encodinghaving one or more different transgenes). In some embodiments, acomposition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more differentrAAVs each having one or more different transgenes.

Suitable carriers may be readily selected by one of skill in the art inview of the indication for which the rAAV is directed. For example, onesuitable carrier includes saline, which may be formulated with a varietyof buffering solutions (e.g., phosphate buffered saline). Otherexemplary carriers include sterile saline, lactose, sucrose, calciumphosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, andwater. The selection of the carrier is not a limitation of the presentdisclosure.

Optionally, the compositions of the disclosure may contain, in additionto the rAAV and carrier(s), other conventional pharmaceuticalingredients, such as preservatives, or chemical stabilizers. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol. Suitable chemical stabilizersinclude gelatin and albumin.

rAAVs are administered in sufficient amounts to transfect the cells of adesired tissue and to provide sufficient levels of gene transfer andexpression without undue adverse effects. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the selected organ (e.g., intraportaldelivery to the liver), oral, inhalation (including intranasal andintratracheal delivery), intraocular, intravenous, intramuscular,subcutaneous, intradermal, intratumoral, and other parental routes ofadministration. Routes of administration may be combined, if desired.

The dose of rAAV virions required to achieve a particular “therapeuticeffect,” e.g., the units of dose in genome copies/per kilogram of bodyweight (GC/kg), will vary based on several factors including, but notlimited to: the route of rAAV virion administration, the level of geneor RNA expression required to achieve a therapeutic effect, the specificdisease or disorder being treated, and the stability of the gene or RNAproduct. One of skill in the art can readily determine a rAAV viriondose range to treat a patient having a particular disease or disorderbased on the aforementioned factors, as well as other factors that arewell known in the art.

An effective amount of an rAAV is an amount sufficient to target infectan animal, target a desired tissue. In some embodiments, an effectiveamount of an rAAV is an amount sufficient to produce a stable somatictransgenic animal model. The effective amount will depend primarily onfactors such as the species, age, weight, health of the subject, and thetissue to be targeted, and may thus vary among animal and tissue. Forexample, an effective amount of the rAAV is generally in the range offrom about 1 ml to about 100 ml of solution containing from about 10⁹ to10¹⁶ genome copies. In some cases, a dosage between about 10¹¹ to 10¹²rAAV genome copies is appropriate. In certain embodiments, 10¹² rAAVgenome copies is effective to target heart, liver, and pancreas tissues.In some cases, stable transgenic animals are produced by multiple dosesof an rAAV.

In some embodiments, rAAV compositions are formulated to reduceaggregation of AAV particles in the composition, particularly where highrAAV concentrations are present (e.g., ˜10¹³ GC/ml or more). Methods forreducing aggregation of rAAVs are well known in the art and, include,for example, addition of surfactants, pH adjustment, salt concentrationadjustment, etc. (See, e.g., Wright F R, et al., Molecular Therapy(2005) 12, 171-178, the contents of which are incorporated herein byreference.)

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 70% or 80% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound in eachtherapeutically-useful composition may be prepared is such a way that asuitable dosage will be obtained in any given unit dose of the compound.Factors such as solubility, bioavailability, biological half-life, routeof administration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

In certain circumstances it will be desirable to deliver the rAAV-basedtherapeutic constructs in suitably formulated pharmaceuticalcompositions disclosed herein either subcutaneously,intrapancreatically, intranasally, parenterally, intravenously,intramuscularly, intrathecally, or orally, intraperitoneally, or byinhalation. In some embodiments, the administration modalities asdescribed in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (eachspecifically incorporated herein by reference in its entirety) may beused to deliver rAAVs. In some embodiments, a preferred mode ofadministration is by portal vein injection.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. Dispersions may also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. In many cases the form issterile and fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art. For example, one dosage may be dissolvedin 1 ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the host. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual host.

Sterile injectable solutions are prepared by incorporating the activerAAV in the required amount in the appropriate solvent with various ofthe other ingredients enumerated herein, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The rAAV compositions disclosed herein may also be formulated in aneutral or salt form. Pharmaceutically-acceptable salts, include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Supplementary active ingredients can also be incorporated into thecompositions. The phrase “pharmaceutically-acceptable” refers tomolecular entities and compositions that do not produce an allergic orsimilar untoward reaction when administered to a host.

Delivery vehicles such as liposomes, nanocapsules, microparticles,microspheres, lipid particles, vesicles, and the like, may be used forthe introduction of the compositions of the present disclosure intosuitable host cells. In particular, the rAAV vector delivered transgenesmay be formulated for delivery either encapsulated in a lipid particle,a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically acceptable formulations of the nucleic acids or therAAV constructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art. Recently, liposomes weredeveloped with improved serum stability and circulation half-times (U.S.Pat. No. 5,741,516). Further, various methods of liposome and liposomelike preparations as potential drug carriers have been described (U.S.Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures. In addition,liposomes are free of the DNA length constraints that are typical ofviral-based delivery systems. Liposomes have been used effectively tointroduce genes, drugs, radiotherapeutic agents, viruses, transcriptionfactors and allosteric effectors into a variety of cultured cell linesand animals. In addition, several successful clinical trials examiningthe effectiveness of liposome-mediated drug delivery have beencompleted.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500.ANG., containing an aqueous solution in the core.

Alternatively, nanocapsule formulations of the rAAV may be used.Nanocapsules can generally entrap substances in a stable andreproducible way. To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 μm) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use.

In addition to the methods of delivery described above, the followingtechniques are also contemplated as alternative methods of deliveringthe rAAV compositions to a host. Sonophoresis (i.e., ultrasound) hasbeen used and described in U.S. Pat. No. 5,656,016 as a device forenhancing the rate and efficacy of drug permeation into and through thecirculatory system. Other drug delivery alternatives contemplated areintraosseous injection (U.S. Pat. No. 5,779,708), microchip devices(U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al.,1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) andfeedback-controlled delivery (U.S. Pat. No. 5,697,899).

Methods of Treating Huntington's Disease

Without wishing to be bound by any particular theory, rAAV comprisingN-terminally grafted heterologous targeting peptides may betherapeutically effective for treating CNS-associated diseases, such asHuntington's disease. Thus, the disclosure relates, in some aspects, tomethods of treating Huntington's disease.

In some embodiments, the disclosure provides a method of treatingHuntington's disease, the method comprising administering to a subjecthaving or suspected of having Huntington's disease an rAAV comprising(i) an N-terminally grafted heterologous targeting peptide and (ii) atransgene encoding an inhibitory RNA targeting a Huntington'sdisease-associated protein. In some embodiments, the N-terminallygrafted heterologous targeting peptide is represented by SEQ ID NO: 7.In some embodiments, the transgene encodes an shRNA or microRNA thathybridizes to and inhibits activity of huntingtin (Htt) protein. In someembodiments, the transgene comprises a nucleic acid having a sequenceset forth in SEQ ID NO: 33. In some embodiments, the transgene encodes aprotein that suppresses or modulates Htt activity or expression. Forexample, in some embodiments, the transgene encodes a zinc fingerprotein that binds specifically to an expanded CAG repeat allele of anHtt gene and suppresses Htt expression. In some embodiments, thetrandgene encodes a protein that modulates protein processing of Htt incells, for example, XBP1.

Kits and Related Compositions

The agents described herein may, in some embodiments, be assembled intopharmaceutical or diagnostic or research kits to facilitate their use intherapeutic, diagnostic or research applications. A kit may include oneor more containers housing the components of the disclosure andinstructions for use. Specifically, such kits may include one or moreagents described herein, along with instructions describing the intendedapplication and the proper use of these agents. In certain embodimentsagents in a kit may be in a pharmaceutical formulation and dosagesuitable for a particular application and for a method of administrationof the agents. Kits for research purposes may contain the components inappropriate concentrations or quantities for running variousexperiments.

In some embodiments, the disclosure relates to a kit for producing arAAV, the kit comprising a container housing an isolated nucleic acidhaving a sequence of any one of SEQ ID NOs: 8-18. In some embodiments,the kit further comprises instructions for producing the rAAV. In someembodiments, the kit further comprises at least one container housing arecombinant AAV vector, wherein the recombinant AAV vector comprises atransgene.

In some embodiments, the disclosure relates to a kit comprising acontainer housing a recombinant AAV having an isolated AAV capsidprotein having an amino acid sequence as set forth in any of SEQ ID NOs:19-27.

The kit may be designed to facilitate use of the methods describedherein by researchers and can take many forms. Each of the compositionsof the kit, where applicable, may be provided in liquid form (e.g., insolution), or in solid form, (e.g., a dry powder). In certain cases,some of the compositions may be constitutable or otherwise processable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species (for example, water or a cell culture medium),which may or may not be provided with the kit. As used herein,“instructions” can define a component of instruction and/or promotion,and typically involve written instructions on or associated withpackaging of the disclosure. Instructions also can include any oral orelectronic instructions provided in any manner such that a user willclearly recognize that the instructions are to be associated with thekit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet,and/or web-based communications, etc. The written instructions may be ina form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, whichinstructions can also reflects approval by the agency of manufacture,use or sale for animal administration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing agents described herein. The agents may bein the form of a liquid, gel or solid (powder). The agents may beprepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include the active agents premixed and shippedin a syringe, vial, tube, or other container. The kit may have one ormore or all of the components required to administer the agents to ananimal, such as a syringe, topical application devices, or iv needletubing and bag, particularly in the case of the kits for producingspecific somatic animal models.

In some cases, the methods involve transfecting cells with totalcellular DNAs isolated from the tissues that potentially harbor proviralAAV genomes at very low abundance and supplementing with helper virusfunction (e.g., adenovirus) to trigger and/or boost AAV rep and cap genetranscription in the transfected cell. In some cases, RNA from thetransfected cells provides a template for RT-PCR amplification of cDNAand the detection of novel AAVs. In cases where cells are transfectedwith total cellular DNAs isolated from the tissues that potentiallyharbor proviral AAV genomes, it is often desirable to supplement thecells with factors that promote AAV gene transcription. For example, thecells may also be infected with a helper virus, such as an Adenovirus ora Herpes Virus. In a specific embodiment, the helper functions areprovided by an adenovirus. The adenovirus may be a wild-type adenovirus,and may be of human or non-human origin, preferably non-human primate(NHP) origin. Similarly adenoviruses known to infect non-human animals(e.g., chimpanzees, mouse) may also be employed in the methods of thedisclosure (See, e.g., U.S. Pat. No. 6,083,716). In addition towild-type adenoviruses, recombinant viruses or non-viral vectors (e.g.,plasmids, episomes, etc.) carrying the necessary helper functions may beutilized. Such recombinant viruses are known in the art and may beprepared according to published techniques. See, e.g., U.S. Pat. No.5,871,982 and U.S. Pat. No. 6,251,677, which describe a hybrid Ad/AAVvirus. A variety of adenovirus strains are available from the AmericanType Culture Collection, Manassas, Va., or available by request from avariety of commercial and institutional sources. Further, the sequencesof many such strains are available from a variety of databasesincluding, e.g., PubMed and GenBank.

Cells may also be transfected with a vector (e.g., helper vector) whichprovides helper functions to the AAV. The vector providing helperfunctions may provide adenovirus functions, including, e.g., E1a, E1b,E2a, E4ORF6. The sequences of adenovirus gene providing these functionsmay be obtained from any known adenovirus serotype, such as serotypes 2,3, 4, 7, 12 and 40, and further including any of the presentlyidentified human types known in the art. Thus, in some embodiments, themethods involve transfecting the cell with a vector expressing one ormore genes necessary for AAV replication, AAV gene transcription, and/orAAV packaging.

In some cases, a novel isolated capsid gene can be used to construct andpackage recombinant AAV vectors, using methods well known in the art, todetermine functional characteristics associated with the novel capsidprotein encoded by the gene. For example, novel isolated capsid genescan be used to construct and package recombinant AAV (rAAV) vectorscomprising a reporter gene (e.g., B-Galactosidase, GFP, Luciferase,etc.). The rAAV vector can then be delivered to an animal (e.g., mouse)and the tissue targeting properties of the novel isolated capsid genecan be determined by examining the expression of the reporter gene invarious tissues (e.g., heart, liver, kidneys) of the animal. Othermethods for characterizing the novel isolated capsid genes are disclosedherein and still others are well known in the art.

The kit may have a variety of forms, such as a blister pouch, a shrinkwrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, ora similar pouch or tray form, with the accessories loosely packed withinthe pouch, one or more tubes, containers, a box or a bag. The kit may besterilized after the accessories are added, thereby allowing theindividual accessories in the container to be otherwise unwrapped. Thekits can be sterilized using any appropriate sterilization techniques,such as radiation sterilization, heat sterilization, or othersterilization methods known in the art. The kit may also include othercomponents, depending on the specific application, for example,containers, cell media, salts, buffers, reagents, syringes, needles, afabric, such as gauze, for applying or removing a disinfecting agent,disposable gloves, a support for the agents prior to administration etc.

The instructions included within the kit may involve methods fordetecting a latent AAV in a cell. In addition, kits of the disclosuremay include, instructions, a negative and/or positive control,containers, diluents and buffers for the sample, sample preparationtubes and a printed or electronic table of reference AAV sequence forsequence comparisons.

EXAMPLES Example 1 Peptide Grafting Yields Novel AAV Vectors Capable ofEnhanced Neuronal Transduction

Several AAV capsids were re-engineered by genetic grafting of differentpeptides into the capsid—naturally occurring AAVrh8, and a liverde-targeted AAV9 mutant, AAV9.47. Table 1 shows a list of there-engineered AAV capsids. Angiopep-2 has been reported to be capable ofcrossing the blood brain barrier by transcytosis. Both capsids toleratedthe insertion of Angiopep-2 (BTP) in the N-terminus of VP2 capsidprotein and the modified capsids were packaged using an approach inwhich VP2 is expressed from one plasmid and VP1 and VP3 are expressedfrom another plasmid at stoichiometric ratios comparable to expressingall three capsid proteins from a single plasmid (FIG. 1A). High titerrecombinant self-complementary vectors encoding GFP under a CBA promoterwere generated in this manner, and western blot analysis confirmed thecorrect stoichiometry of all three capsid proteins (FIG. 1B).

TABLE 1 List of peptides inserted in VP2 of AAVrh8 or AAV9.47 Effect SEQParental on CNS ID Peptide capsid tropism Peptide amino acid sequenceNO. FC5 AAVrh8 Reduce EVQLQASGGGLVQAGGSLRLSCAAS 1GFKITHYTMGWFRQAPGKEREFVSR ITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYCAAG STSTATPLRVDYWGKGTQVTVSS FC44 AAVrh8 ReduceEVQLQASGGGLVQAGGSLRLSCSAS 2 VRTFSIYAMGWFRQAPGKEREFVAGINRSGDVTKYADFVKGRFSISRDNA KNMVYLQMNSLKPEDTALYYCAATWAYDTVGALTSGYNFWGQGTQVT VSS ApoB100 AAVrh8 No effectPSSVIDALQYKLEGTTRLTRKRGLKL 3 ATALSLSNKFVEGSPS RVG AAVrh8 No effectYTIWMPENPRPGTPCDIFTNSRGKRA 4 SNG Angiopep- AAVrh8 IncreaseTFFYGGSRGKRNNFKTEEY 5 2 ICAMg3 AAV9.47 No effect NNQKIVNLKEKVAQLEA 6 RVGAAV9.47 No effect YTIWMPENPRPGTPCDIFTNSRGKRA 4 SNG Angiopep- AAV9.47Increase TFFYGGSRGKRNNFKTEEY 5 2 A- AAV9.47 Increase AAAAAAAAAAAAAAAAAAA7 string

The biodistribution of the two re-engineered vectors was assessed bysystemic infusion via tail vein in 6-8week-old C57BL/6 mice at a dose of5E11 vg. Four weeks post-infusion, significant increase in CNStransduction was observed for both re-engineered capsids compared to theparent capsids (FIG. 2). Both new AAV vectors transduced brainendothelium, glia and cortical neurons at exceptional high efficiency.Insertion of other brain penetrating peptides, e.g., RVG, ICAMg3,ApoB100, etc. in the context of either parent capsid did not lead toincreased CNS transduction at levels comparable to angiopep-2.Remarkably, different neuronal populations were targeted by the samepeptide in different capsid backgrounds. The re-engineered AAVrh8 vectortransduced thalamic neurons and neurons in the visual cortex, whileneurons in the striatum were transduced only by the re-engineeredAAV9.47 vector (Fig.3). This indicates that the enhanced neural tropismof these new AAV vectors is a function of the interplay between peptideand capsid. Transduction of thalamic and striatal neurons was notobserved upon dose-matched administration of the native capsids or AAV9.Importantly, no increase in transduction of non-target peripheraltissues was observed with the re-engineered capsids (FIG. 4).

Unexpectedly, the insertion of a 19-amino acid alanine “string” (AS) inthe VP2 capsid protein of AAV9.47 further increased CNS tropism comparedto the Angiopep-2-carrying counterpart. Cortical and striatal neuronswere transduced in the brain at an unprecedented efficiency, while motorneurons and interneurons were transduced in the spinal cord (FIG. 5).Significantly more vector genomes were found in the cerebrum and spinalcord of AS-grafted rAAV9.47 (rAAV9.47-AS), compared to AAV9 (15-fold and6-fold, respectively) (FIG. 6). Importantly, no significant differencein vector genomes was observed in non-target peripheral tissues (liver,skeletal muscle, heart, lung, kidney, pancreas and spleen) betweenrAAV9.47-AS and AAV9 (FIG. 7).

Example 2 Widespread CNS Gene Transfer and Silencing after SystemicDelivery of a Novel AAV Vector

Effective gene delivery to the central nervous system (CNS) is vital fordevelopment of novel gene therapies for neurological diseases.Adeno-associated virus (AAV) vectors have emerged as an effectiveplatform for in vivo gene transfer, but overall neuronal transductionefficiency of vectors derived from naturally occurring AAV capsids aftersystemic administration is relatively low. Here, the possibility ofenhancing CNS transduction of existing AAV capsids by genetically fusingpeptides to the N-terminus of VP2 capsid protein was investigated. Anovel vector AAV-AS, generated by the insertion of a poly-alaninepeptide, is capable of extensive gene transfer throughout the CNS aftersystemic administration in adult mice. AAV-AS transduced 36% of striatalneurons, which is 80-fold higher than AAV9. Widespread neuronaltransduction was also documented in cat brain after systemic infusion ofAAV-AS. A single intravenous injection of an AAV-AS vector encoding anartificial microRNA targeting huntingtin (Htt) resulted in 40-50%knockdown of Htt across multiple CNS structures in adult mice.

Materials and Methods Generation of Packaging Constructs

AAV9.47 trans packaging plasmid was generated by replacing a portion ofthe AAV9 cap sequence in packaging plasmid pAR-9 with a de-novosynthesized fragment carrying the following mutations S414N, G453D,K557E and T582I (GenScript USA Inc., Piscataway, N.J.) (amino acidnumbering beginning at VP1) using In-Fusion cloning kit (ClontechLaboratories Inc., Mountain View, Calif.). Packaging plasmid necessaryto express either only VP1 and VP3 capsid proteins, or VP2 protein fusedto peptide were generated by introducing point mutations (T138Asubstitution to generate VP1,3 plasmids; MIL for VP2 plasmids) asdescribed for AAV2. Peptide and linker (G₄5) coding sequences werecloned at the N-terminus of VP2 using In-Fusion cloning kit to generatepeptide-VP2 expression plasmids.

Vector Particle Production, Titer Quantification and Quality Analysis

The self-complementary AAV-CBA-GFP vector used in these studies carriesan expression cassette comprised of the CBA promoter without an intronto drive expression of GFP and a rabbit β-globin poly-adenylationsignal. Sequences targeting mouse Hu mRNA were embedded into theartificial miR-155 scaffold to generate the following cassette:5′-ctggaggcttgctgaaggctgtatgctgTTTAGACTTGTGTCCTTGACCTgttttggccactgactgacTGGCAAAGCACAAGTCTAAAcaggacacaaggcctgttactagcactcacatggaacaaatggcc-3′ (SEQID NO: 33; targeting sequence in bold uppercase). This artificial miRNAtargets position 1090 in exon 8 of mouse huntingtin gene. eGFP andartificial miRNA cassette were expressed under the control of thecytomegalovirus enhancer/chicken β-actin promoter (CBA) containing theβ-actin exon and chimeric intron.

To generate peptide-grafted AAV vectors, 293T cells were co-transfectedwith the following mix of plasmids using the calcium phosphateprecipitation method: 7.96 μg transgene plasmid, 25.6 μg adenoviralhelper plasmid pFΔ6, and a 5:1 ratio of VP1,3 packaging plasmid andpeptide-VP2 packaging fused expression plasmid in trans, for a totalamount of 12.2 μg, per 2.1×10⁷ cells plated. 72 hours post transfection,cells were harvested and cell lysates prepared by 3 cycles offreeze-thaw and treated with Benzonase (Sigma-Aldrich, St. Louis, Mo.)(50 U/mL cell lysate, 37° C., 30 min). rAAV was purified from celllysates by iodixanol density gradient ultracentrifugation (Optiprepdensity gradient medium, Axis-Shield, Oslo, Norway). Residual iodixanolwas removed by replacing with Buffer B (20 mM TRIS, 0.5 M NaCl, pH 8.5)using a 100 kilodalton (kDa) cutoff centrifugation device (AmiconUltra-15, Merck Millipore Ltd., Cork, Ireland) by three rounds ofcentrifugation at 1500× g and dialyzed twice using a 10,000 molecularweight cutoff (MWCO) dialysis cassette (Slide-A-Lyzer, ThermoScientific, Rockford, Ill.) against a 1,000-fold volume of PBS for >2 hand once overnight at 4° C. After treatment of stocks with DNase I(Roche Diagnostics GmbH, Mannheim, Germany, 2 U/μL vector, 37° C., 30min), the titer of rAAV vectors was determined by real-time quantitativePCR (qPCR) using probe and primers specific for the rabbit 13-globinpolyA sequence (Integrated DNA Technologies, Coralville, Iowa). Forstoichiometric analysis of capsid proteins, 1×10¹⁰ vector particles ofpurified vector were subjected to Western blotting by standard SDS-PAGEtechnique. AAV capsid proteins were detected using mouse monoclonalanti-AAV capsid protein antibody clone B1 (1:500, American ResearchProducts, Inc., Waltham, Mass., 03-65158), peroxidase linked anti-mousesecondary antibody (1:2000, GE Healthcare UK Ltd., Buckinghamshire, UK,380199) and ECL Western Blotting Substrate (Pierce Protein ResearchProducts, Rockford, Ill.).

Vector Administration and Tissue Processing

rAAV vectors were administered via the tail vein in a volume of 200 μLinto 6-8 week-old male C57BL/6J mice (Jackson Laboratory, Bar Harbor,Me.). A dose of 5×10¹¹ vg/mouse was administered for immunochemicalstudies and biodistribution analysis. For immunochemical and GFPfluorescence studies, mice were trans-cardially perfused at 4 weekspost-injection first with ice cold 1× phosphate buffer saline (PBS),followed by 10% phosphate-buffered formalin solution (Fisher Scientific,Fair Lawn, N.J.). Tissues were harvested and post-fixed in 10%phosphate-buffered formalin solution at 4° C. for an additional 24 h.Post-fixed tissues were transferred to 30% sucrose in 1× PBS forcryoprotection. Tissues were embedded in Tissue-Tek O.C.T. compound(Sakura Finetek, Torrance, Calif.) and frozen in a dry-ice-isopentanebath and stored at −80° C.

For biodistribution analysis, mice were trans-cardially perfused at 4weeks post-infusion with ice-cold 1× PBS. Tissues were harvestedimmediately, frozen on dry ice and stored at −80° C.

Cat Studies

rAAV vector was packaged by University of Massachusetts Medical SchoolViral Vector Core by transient transfection followed by purification bycesium chloride sedimentation, and administered through the carotidartery into a 2 month old normal domestic short haired cat at a dose of1.29×10¹³ vg. At 4 weeks post infusion, the injected cat wastrans-cardially perfused with cold 1× PBS. Various tissues wereharvested and fixed in 4% paraformaldehyde in PBS at 4° C. The brain wascut into 0.6 cm coronal blocks prior to immersion in fixative.Processing of post-fixed brains and spinal cords for immunohistochemical(IHC) and immunofluorescence studies was identical to that for mousestudies.

Immunohistochemical Detection of GFP Expression

For chromogenic IHC, 40 μm serial sections of brains and 30 μm serialsections of spinal cord were incubated for 96 h in anti-GFP primaryantibody (ABfinity rabbit monoclonal anti-GFP 1:1000, G10362, LifeTechnologies, Grand Island, N.Y.) at 4° C. After washing with 1× PBS,sections were incubated in appropriate biotinylated secondary antibody(biotinylated anti-rabbit antibody, Vector Laboratories Inc.,Burlingame, Calif.), followed by incubation in ABC reagent (PK-6100,Vector Laboratories Inc.). Sections were developed with3,3′-diaminobenzidine reagent (DAB) according to the manufacturer'sinstructions (SK-4100, Vector Laboratories Inc.), dehydrated withincreasing concentrations of ethanol, cleared with xylene and mountedusing Permount mounting medium (Fisher Scientific).

For immunofluorescence studies, 40 μm sections of brains and 30 μmsections of spinal cord were incubated for 24 h in a cocktail ofappropriate primary antibodies at 4° C. The primary antibodies usedwere: rabbit polyclonal anti-GFP (1:1000, Life Technologies, A11122),mouse monoclonal anti-NeuN (1:500, EMD Millipore, MAB377), mousemonoclonal anti-DARPP32 (1:250, BD Biosciences, 611520). After washingin 1× PBS, sections were incubated for 1 h at room temperature inappropriate secondary antibodies, washed in 1× PBS and mounted usingPermafluor mounting media (Thermo Scientific). Native GFP fluorescencein liver and skeletal muscle (quadriceps) was analyzed in 30 μm sectionsmounted using Permafluor mounting media. All images were captured on aLeica DM5500 B microscope (Leica Microsystems Inc., Buffalo Grove,Ill.). Post-processing of images was performed using Adobe Photoshop CS6(Adobe Systems, San Jose, Calif.).

Quantification of GFP-Positive Neurons in Striatum and Thalamus

Chromogenic IHC staining of 40 μm mouse brain sections was performed.Five 663.28 μm×497.40 μm regions were randomly chosen from the striatumor thalamus (n=4 biological replicates per vector) of the stainedsections. Neurons were identified by their morphology and counted byindividuals blinded to the study design. All statistical analyses wereperformed using GraphPad Prism (GraphPad Software, Inc., La Jolla,Calif.). Total neurons in the 663.28 μm×497.40 μm fields were counted inNissl (cresyl violet acetate) stained brain sections using ImageJsoftware. Significance was determined by Student's unpaired T-test. Ap<0.05 was considered to be significant.

Biodistribution Analysis

Vector genome copy numbers from various mouse tissues were determined byqPCR after extraction of total DNA using DNeasy Blood and Tissue kit(Qiagen). Tissues were mechanically lysed using TissueLyzer II (QiagenGmbH, Hilden, Germany). Vector genome content in each tissue wasdetermined using 100 ng total DNA using the same qPCR method describedabove for AAV vector titration. All statistical analyses were performedusing GraphPad Prism. Significance was determined by Student's unpairedT-test. A p<0.05 was considered to be significant.

Western blotting to detect GFP protein levels in various tissue typeswas performed using primary antibodies detecting GFP (chicken polyclonalanti-GFP, 1:2000, Ayes Labs Inc., Tigard, Oreg., GFP-1010) and mouseβ-actin (mouse monoclonal anti-β-actin, 1:1000, Sigma-Aldrich, St.Louis, Mo., A5441), followed by appropriate IRDye secondary antibodies(LI-COR Inc., Lincoln, Nebr.). Total protein was isolated from harvestedtissues by bead lysis in T-PER tissue extraction reagent (LifeTechnologies) and quantified by Bradford assay. 20 μg of total proteinwas loaded onto each well of 4-20% Mini-PROTEAN TGX gels (Bio-RadLaboratories Inc., Hercules, Calif.). Tissues from two representativemice per group were used for analysis. Detection and quantification weredone with Odyssey infrared imaging system (LI-COR Inc.).

In-Vitro Binding Assay

ProS and Lec2 CHO cell lines were gifts from Dr. Aravind Asokan(University of North Carolina Chapel Hill) and binding assay wasperformed as previously described. Briefly, cells were pre-chilled for30 min at 4° C. in serum-free DMEM (Life Technologies), followed byincubation with rAAV vectors at 4×10⁴ vg/cell in cold serum-free mediaDMEM at 4° C. 90 min later, cells were washed thrice with coldserum-free DMEM to remove loosely bound vector particles. Cells wereharvested and total DNA was extracted using DNeasy Blood and Tissue kit(Qiagen). Vector genome copy numbers of cell surface bound virions wasquantified by qPCR.

Analysis of Hit Knockdown

rAAV vectors were administered intravascularly via the tail vein into6-8 week-old male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) ata dose of 9.4×10¹¹ vg/mouse. Mice were euthanized at 4 weekspost-injection and the brain sectioned in 2 mm coronal blocks using abrain matrix. Biopsy punches of different diameters (2 or 3 mm) wereused to sample motor cortex (2 mm), striatum (3 mm) and thalamus (3 mm).Cervical spinal cord and liver were also included in the analysis.Tissue samples were mechanically homogenized using a TissueLyzer II and5 mm stainless steel beads (Qiagen) in Trizol (Life Technologies). TotalRNA was isolated using Direct-zol RNA MiniPrep kit (Zymo ResearchCorporation, Irvine, Calif.) according to manufacturer's protocol. TotalRNA (400-1000 ng) was reverse transcribed using High Capacity RNA tocDNA kit (Applied Biosystems, Foster City, Calif.). Relative mouse HttmRNA expression was assessed by qPCR using TaqMan gene expression assaysfor mouse Htt (Mm01213820_ml, Applied Biosystems) and hypoxanthinephosphoribosyltransferase 1 (Hprt1; mm00446968_ml, Applied Biosystems).Changes in Htt mRNA for groups injected with AAV9 or AAV-AS vectors werecalculated relative to PBS-injected mice using the 2^(−ΔΔCT) method.Significance was determined by Student's unpaired T-test. A p<0.05 wasconsidered to be significant.

For protein analysis, a frozen punch from each region (motor cortex,striatum, thalamus, cervical spinal cord, and liver) was homogenized in75-300 μl 10 mM HEPES pH7.4, 250 mM sucrose, 1 mM EDTA+proteaseinhibitor tablet (cOmplete mini, EDTA-free, Roche), 1 mM NaF and 1 mMNa₃VO₄ on ice for 30 strokes. Protein concentration was determined byBradford method (BioRad) and 10 μg motor cortex, striatum, and thalamusor 20 μg cervical spinal cord and liver were loaded onto 3-8%Tris-Acetate gels (Life Technologies) and separated by SDS-PAGE.Proteins were transferred to nitrocellulose using a TransBlot Turboapparatus (BioRad) then blots were cut horizontally at 72 kD. Blots werewashed in TRIS-buffered saline +0.1% Tween 20 (TBST) and blocked in 5%milk/TBST. The top half of the blot was incubated in anti-Htt antibodyAb1 (1:2000) and the bottom half in anti-tubulin antibody (1:4000,Sigma) or anti-GAPDH antibody (1:6000, Millipore) diluted in 5%milk/TBST overnight at 4° C. Blots were washed in TBST then incubated inperoxidase conjugated secondary antibodies diluted in 5% milk/TBST for 1hour at room temperature, washed, and proteins were detected usingSuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) andFluoroChem SP (Alpha Innotech) and Hyperfilm ECL. The bottom blots werereprobed with anti-GFP antibody (1:3000, Cell Signaling). Densitometrywas performed using ImageJ software.

Insertion of Poly-Alanine Peptide in AAV9.47 Capsid Enhances NeuronalGene Transfer in Adult Mice

AAV9.47 was selected as the capsid in which to test the peptide graftingapproach, as this quadruple mutant of AAV9 (S414N, G453D, K557E, T582I)has comparable CNS gene transfer properties to AAV9 but decreasedtropism to liver (FIG. 12). Peptides were grafted on the AAV9.47 capsidsurface via genetic fusion to the N-terminus of VP2 (FIG. 13). Apeptide-modified AAV9.47 vector carrying a string of 19 alanines in theVP2 capsid protein, designated as AAV-AS, showed a remarkable increasein CNS transduction efficiency compared to AAV9 (FIG. 8A) after systemicdelivery in 6-8 week old C57BL/6 mice (FIG. 8A). AAV-AS vectortransduced diverse neuronal populations, glia and endothelia throughoutthe brain and spinal cord, including extensive transduction of neuronsin motor cortex and striatum (FIG. 8B). Efficient transduction ofgranule cells in the dentate gyrus, as well as motor neurons andinterneurons in the spinal cord, was observed (FIG. 8B). The identity ofGFP-positive cells with neuronal morphology in cortex, striatum andspinal cord was confirmed by co-localization of GFP and NeuN (FIG. 8C).GFP-positive neurons in the striatum were shown to be DARPP32-positivemedium spiny neurons (FIG. 8C). Neuronal transduction was apparent inmany brain regions of AAV-AS injected mice with the exception ofthalamus and hypothalamus where only sparse transduction was observed(FIG. 14). AAV-AS also transduced oligodendrocytes in the corpuscallosum and Bergmann glia in the cerebellum (FIG. 14). The transductionprofile of AAV9 was limited to glial cells and endothelia in most CNSregions analyzed (FIG. 8B and FIG. 14).

Western blot analysis of AAV preparations confirmed incorporation of thechimeric poly-alanine VP2 protein in the AAV-AS capsid (FIG. 9A).Quantification of GFP-positive neurons in thalamus and striatum revealeda modest 2-fold increase in transduced thalamic neurons but a striking80-fold increase in transduced striatal neurons for AAV-AS compared toAAV9 (FIG. 9B). AAV-AS transduced as many as 36% of striatal neuronscompared to only 0.45% by AAV9 (FIG. 9B). The CNS transductionefficiency of AAV-AS vector was also reflected in its biodistributionprofile. More vector genomes were found in the cerebrum and spinal cordof AAV-AS injected animals compared to AAV9 (15-fold and 6-fold,respectively) (FIG. 9C). These findings were corroborated by comparableincrease in GFP protein in cerebrum and spinal cord (FIG. 9D). Theincreased gene transfer efficiency for AAV-AS vector compared to AAV9appears to be restricted to CNS, as transduction of liver and muscle wasidentical for both AAV vectors based on analysis of vector genomecontent (FIG. 9E) and GFP protein levels (FIG. 9F).

As a first step to understand how the poly-alanine peptide enhances CNStransduction of AAV9.47, a cell culture study was carried out todetermine whether it changes the capsid interaction with exposedgalactose residues on cell surface glycans. Terminal N-linked galactoseis the primary cell surface receptor for AAV9. Binding studies inparental (Pro5) and sialic acid-deficient (Lec2) CHO cells showed nodifferences between AAV-AS, AAV9.47 and AAV9 vectors (FIG. 15).

AAV-AS Transduces Neurons Throughout the Cat Brain after SystemicAdministration

Next, whether the neuronal transduction properties of AAV-AS arereproducible in cats was assessed. Consistent with results in mice,AAV-AS transduced diverse neuronal populations across the cat brain andspinal cord, including neurons in cerebral cortex, striatum andreticular formation, Purkinje neurons in cerebellum and motor neurons inthe oculomotor nucleus located in ventral midbrain, spinal nucleus ofthe trigeminal nerve in brainstem and throughout the spinal cord (FIG.10A-10B). Curiously, no endothelial and only sparse glial transductionwas apparent in the cat brain in this study.

Widespread Knockdown of Htt in CNS after Systemic Delivery of AAV-ASVector

The therapeutic potential of AAV-AS vector for Huntington's disease wasexamined. AAV-AS and AAV9 vectors encoding GFP and an U6-drivenartificial microRNA specific for mouse Htt (miR^(Htt)) were infusedsystemically in C57BL/6 mice. Huntingtin mRNA and protein levels in CNSand liver were assessed at 4 weeks post injection (FIG. 11). AAV-ASvector resulted in 40-50% reduction in Htt mRNA in striatum, motorcortex and spinal cord, and was better than AAV9 in all brain regions,but comparable in the spinal cord (FIG. 11A). Conversely, AAV-AS wasless potent than AAV9 in lowering Hu mRNA in liver (FIG. 11A). Westernblot analysis of Htt protein levels corroborated the differences betweenAAV9 and AAV-AS in the striatum, motor cortex and thalamus as well as inliver (FIG. 11B). The reduction in Htt protein levels was inverselyproportional to GFP protein levels, which indicates that higher CNStransduction efficiency leads to greater reduction in huntingtin mRNAand protein.

SEQUENCESSEQ ID NO: 8 AAVrh8VP1,3 nucleotide sequence (supplies VP1 and VP3 intrans to peptide-inserted VP2):ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGGCGGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCTCGGGCATCGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCCGATCCACAACCTCTCGGAGAACCTCCAGCAGCCCCCTCAGGTCTGGGACCTAATACAATGGCTTCAGGCGGTGGCGCTCCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGCACCTCGGGAGGAAGCACCAACGACAACACCTATTTTGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGTCACTTTTCACCACGTGACTGGCAACGACTCATCAACAACAATTGGGGATTCCGGCCCAAAAGACTCAACTTCAAGCTGTTCAACATCCAGGTCAAGGAAGTCACGACGAACGAAGGCACCAAGACCATCGCCAATAATCTCACCAGCACCGTGCAGGTCTTTACGGACTCGGAGTACCAGTTACCGTACGTGCTAGGATCCGCTCACCAGGGATGTCTGCCTCCGTTCCCGGCGGACGTCTTCATGGTTCCTCAGTACGGCTATTTAACTTTAAACAATGGAAGCCAAGCCCTGGGACGTTCCTCCTTCTACTGTCTGGAGTATTTCCCATCGCAGATGCTGAGAACCGGCAACAACTTTCAGTTCAGCTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCCTGGACAGGCTGATGAATCCCCTCATCGACCAGTACCTGTACTACCTGGTCAGAACGCAAACGACTGGAACTGGAGGGACGCAGACTCTGGCATTCAGCCAAGCGGGTCCTAGCTCAATGGCCAACCAGGCTAGAAATTGGGTGCCCGGACCTTGCTACCGGCAGCAGCGCGTCTCCACGACAACCAACCAGAACAACAACAGCAACTTTGCCTGGACGGGAGCTGCCAAGTTTAAGCTGAACGGCCGAGACTCTCTAATGAATCCGGGCGTGGCAATGGCTTCCCACAAGGATGACGACGACCGCTTCTTCCCTTCGAGCGGGGTCCTGATTTTTGGCAAGCAAGGAGCCGGGAACGATGGAGTGGATTACAGCCAAGTGCTGATTACAGATGAGGAAGAAATCAAGGCTACCAACCCCGTGGCCACAGAAGAATATGGAGCAGTGGCCATCAACAACCAGGCCGCCAATACGCAGGCGCAGACCGGACTCGTGCACAACCAGGGGGTGATTCCCGGCATGGTGTGGCAGAATAGAGACGTGTACCTGCAGGGTCCCATCTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCGTCTCCCCTGATGGGCGGCTTTGGACTGAAGCACCCGCCTCCTCAAATTCTCATCAAGAACACACCGGTTCCAGCGGACCCGCCGCTTACCTTCAACCAGGCCAAGCTGAACTCTTTCATCACGCAGTACAGCACCGGACAGGTCAGCGTGGAAATCGAGTGGGAGCTGCAGAAAGAAAACAGCAAACGCTGGAATCCAGAGATTCAATACACTTCCAACTACTACAAATCTACAAATGTGGACTTTGCTGTCAACACGGAGGGGGTTTATAGCGAGCCTCGCCCCATTGGCACCCGTTACCTCACCCGCAACCTGTAASEQ ID NO: 9 AAVrh8VP2FC5 nucleotide sequence (supplies FC5 insertedVP2):(Underlined sequence indicates peptide and conjugated linker sequence)CTGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGATGGAAGTGCAGCTCCAAGCCAGTGGTGGCGGACTGGTGCAGGCAGGAGGCAGCTTGAGGCTCAGCTGTGCCGCCAGCGGCTTCAAGATCACCCACTACACCATGGGCTGGTTCCGGCAGGCCCCTGGCAAGGAGAGGGAGTTCGTGAGCAGGATTACCTGGGGTGGTGACAACACCTTCTACAGCAACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGACTACTACTGTGCAGCCGGCAGTACCAGCACCGCTACCCCCCTGAGGGTGGACTACTGGGGCAAAGGCACTCAGGTGACAGTGTCTTCAGGAGGGGGTGGCAGCGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCTCGGGCATCGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCCGATCCACAACCTCTCGGAGAACCTCCAGCAGCCCCCTCAGGTCTGGGACCTAATACAATGGCTTCAGGCGGTGGCGCTCCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGCACCTCGGGAGGAAGCACCAACGACAACACCTATTTTGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGTCACTTTTCACCACGTGACTGGCAACGACTCATCAACAACAATTGGGGATTCCGGCCCAAAAGACTCAACTTCAAGCTGTTCAACATCCAGGTCAAGGAAGTCACGACGAACGAAGGCACCAAGACCATCGCCAATAATCTCACCAGCACCGTGCAGGTCTTTACGGACTCGGAGTACCAGTTACCGTACGTGCTAGGATCCGCTCACCAGGGATGTCTGCCTCCGTTCCCGGCGGACGTCTTCATGGTTCCTCAGTACGGCTATTTAACTTTAAACAATGGAAGCCAAGCCCTGGGACGTTCCTCCTTCTACTGTCTGGAGTATTTCCCATCGCAGATGCTGAGAACCGGCAACAACTTTCAGTTCAGCTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCCTGGACAGGCTGATGAATCCCCTCATCGACCAGTACCTGTACTACCTGGTCAGAACGCAAACGACTGGAACTGGAGGGACGCAGACTCTGGCATTCAGCCAAGCGGGTCCTAGCTCAATGGCCAACCAGGCTAGAAATTGGGTGCCCGGACCTTGCTACCGGCAGCAGCGCGTCTCCACGACAACCAACCAGAACAACAACAGCAACTTTGCCTGGACGGGAGCTGCCAAGTTTAAGCTGAACGGCCGAGACTCTCTAATGAATCCGGGCGTGGCAATGGCTTCCCACAAGGATGACGACGACCGCTTCTTCCCTTCGAGCGGGGTCCTGATTTTTGGCAAGCAAGGAGCCGGGAACGATGGAGTGGATTACAGCCAAGTGCTGATTACAGATGAGGAAGAAATCAAGGCTACCAACCCCGTGGCCACAGAAGAATATGGAGCAGTGGCCATCAACAACCAGGCCGCCAATACGCAGGCGCAGACCGGACTCGTGCACAACCAGGGGGTGATTCCCGGCATGGTGTGGCAGAATAGAGACGTGTACCTGCAGGGTCCCATCTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCGTCTCCCCTGATGGGCGGCTTTGGACTGAAGCACCCGCCTCCTCAAATTCTCATCAAGAACACACCGGTTCCAGCGGACCCGCCGCTTACCTTCAACCAGGCCAAGCTGAACTCTTTCATCACGCAGTACAGCACCGGACAGGTCAGCGTGGAAATCGAGTGGGAGCTGCAGAAAGAAAACAGCAAACGCTGGAATCCAGAGATTCAATACACTTCCAACTACTACAAATCTACAAATGTGGACTTTGCTGTCAACACGGAGGGGGTTTATAGCGAGCCTCGCCCCATTGGCACCCGTTACCTCACCCGCAACCTGTAASEQ ID NO: 10 AAVrh8VP2FC44 nucleotide sequence (supplies FC44inserted VP2):(Underlined sequence indicates peptide and conjugated linker sequence)CTGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGATGGAGGTGCAGCTGCAGGCTTCTGGAGGAGGACTGGTGCAGGCTGGAGGGAGTCTGAGGCTGTCTTGCAGTGCCTCAGTGAGGACTTTCTCCATCTACGCCATGGGCTGGTTTAGGCAGGCTCCCGGGAAGGAGCGCGAATTCGTGGCCGGAATCAACCGGTCTGGCGACGTGACCAAGTACGCTGATTTCGTGAAAGGCCGGTTTAGCATTTCCAGAGACAACGCCAAGAATATGGTGTATCTGCAGATGAACTCCCTGAAACCTGAAGACACAGCTCTGTACTATTGTGCCGCTACTTGGGCCTACGATACCGTGGGGGCTCTGACATCAGGATATAATTTTTGGGGCCAGGGGACCCAGGTGACAGTGAGCTCCGGAGGAGGAGGAAGCGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCTCGGGCATCGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCCGATCCACAACCTCTCGGAGAACCTCCAGCAGCCCCCTCAGGTCTGGGACCTAATACAATGGCTTCAGGCGGTGGCGCTCCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGCACCTCGGGAGGAAGCACCAACGACAACACCTATTTTGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGTCACTTTTCACCACGTGACTGGCAACGACTCATCAACAACAATTGGGGATTCCGGCCCAAAAGACTCAACTTCAAGCTGTTCAACATCCAGGTCAAGGAAGTCACGACGAACGAAGGCACCAAGACCATCGCCAATAATCTCACCAGCACCGTGCAGGTCTTTACGGACTCGGAGTACCAGTTACCGTACGTGCTAGGATCCGCTCACCAGGGATGTCTGCCTCCGTTCCCGGCGGACGTCTTCATGGTTCCTCAGTACGGCTATTTAACTTTAAACAATGGAAGCCAAGCCCTGGGACGTTCCTCCTTCTACTGTCTGGAGTATTTCCCATCGCAGATGCTGAGAACCGGCAACAACTTTCAGTTCAGCTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCCTGGACAGGCTGATGAATCCCCTCATCGACCAGTACCTGTACTACCTGGTCAGAACGCAAACGACTGGAACTGGAGGGACGCAGACTCTGGCATTCAGCCAAGCGGGTCCTAGCTCAATGGCCAACCAGGCTAGAAATTGGGTGCCCGGACCTTGCTACCGGCAGCAGCGCGTCTCCACGACAACCAACCAGAACAACAACAGCAACTTTGCCTGGACGGGAGCTGCCAAGTTTAAGCTGAACGGCCGAGACTCTCTAATGAATCCGGGCGTGGCAATGGCTTCCCACAAGGATGACGACGACCGCTTCTTCCCTTCGAGCGGGGTCCTGATTTTTGGCAAGCAAGGAGCCGGGAACGATGGAGTGGATTACAGCCAAGTGCTGATTACAGATGAGGAAGAAATCAAGGCTACCAACCCCGTGGCCACAGAAGAATATGGAGCAGTGGCCATCAACAACCAGGCCGCCAATACGCAGGCGCAGACCGGACTCGTGCACAACCAGGGGGTGATTCCCGGCATGGTGTGGCAGAATAGAGACGTGTACCTGCAGGGTCCCATCTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCGTCTCCCCTGATGGGCGGCTTTGGACTGAAGCACCCGCCTCCTCAAATTCTCATCAAGAACACACCGGTTCCAGCGGACCCGCCGCTTACCTTCAACCAGGCCAAGCTGAACTCTTTCATCACGCAGTACAGCACCGGACAGGTCAGCGTGGAAATCGAGTGGGAGCTGCAGAAAGAAAACAGCAAACGCTGGAATCCAGAGATTCAATACACTTCCAACTACTACAAATCTACAAATGTGGACTTTGCTGTCAACACGGAGGGGGTTTATAGCGAGCCTCGCCCCATTGGCACCCGTTACCTCACCCGCAACCTGTAASEQ ID NO: 11 AAVrh8VP2ApoB100 nucleotide sequence (supplies ApoB100inserted VP2):(Underlined sequence indicates peptide and conjugated linker sequence)CTGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGATGCCAAGCAGTGTAATCGATGCGCTGCAGTACAAGCTGGAGGGCACCACGAGGCTGACCAGGAAGAGGGGTCTGAAGCTGGCCACGGCCCTCAGCCTTAGCAATAAGTTCGTAGAGGGCAGCCCCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCTCGGGCATCGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCCGATCCACAACCTCTCGGAGAACCTCCAGCAGCCCCCTCAGGTCTGGGACCTAATACAATGGCTTCAGGCGGTGGCGCTCCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGCACCTCGGGAGGAAGCACCAACGACAACACCTATTTTGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGTCACTTTTCACCACGTGACTGGCAACGACTCATCAACAACAATTGGGGATTCCGGCCCAAAAGACTCAACTTCAAGCTGTTCAACATCCAGGTCAAGGAAGTCACGACGAACGAAGGCACCAAGACCATCGCCAATAATCTCACCAGCACCGTGCAGGTCTTTACGGACTCGGAGTACCAGTTACCGTACGTGCTAGGATCCGCTCACCAGGGATGTCTGCCTCCGTTCCCGGCGGACGTCTTCATGGTTCCTCAGTACGGCTATTTAACTTTAAACAATGGAAGCCAAGCCCTGGGACGTTCCTCCTTCTACTGTCTGGAGTATTTCCCATCGCAGATGCTGAGAACCGGCAACAACTTTCAGTTCAGCTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCCTGGACAGGCTGATGAATCCCCTCATCGACCAGTACCTGTACTACCTGGTCAGAACGCAAACGACTGGAACTGGAGGGACGCAGACTCTGGCATTCAGCCAAGCGGGTCCTAGCTCAATGGCCAACCAGGCTAGAAATTGGGTGCCCGGACCTTGCTACCGGCAGCAGCGCGTCTCCACGACAACCAACCAGAACAACAACAGCAACTTTGCCTGGACGGGAGCTGCCAAGTTTAAGCTGAACGGCCGAGACTCTCTAATGAATCCGGGCGTGGCAATGGCTTCCCACAAGGATGACGACGACCGCTTCTTCCCTTCGAGCGGGGTCCTGATTTTTGGCAAGCAAGGAGCCGGGAACGATGGAGTGGATTACAGCCAAGTGCTGATTACAGATGAGGAAGAAATCAAGGCTACCAACCCCGTGGCCACAGAAGAATATGGAGCAGTGGCCATCAACAACCAGGCCGCCAATACGCAGGCGCAGACCGGACTCGTGCACAACCAGGGGGTGATTCCCGGCATGGTGTGGCAGAATAGAGACGTGTACCTGCAGGGTCCCATCTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCGTCTCCCCTGATGGGCGGCTTTGGACTGAAGCACCCGCCTCCTCAAATTCTCATCAAGAACACACCGGTTCCAGCGGACCCGCCGCTTACCTTCAACCAGGCCAAGCTGAACTCTTTCATCACGCAGTACAGCACCGGACAGGTCAGCGTGGAAATCGAGTGGGAGCTGCAGAAAGAAAACAGCAAACGCTGGAATCCAGAGATTCAATACACTTCCAACTACTACAAATCTACAAATGTGGACTTTGCTGTCAACACGGAGGGGGTTTATAGCGAGCCTCGCCCCATTGGCACCCGTTACCTCACCCGCAACCTGTAASEQ ID NO: 12 AAVrh8VP2RVG nucleotide sequence (supplies RVG insertedVP2):(Underlined sequence indicates peptide and conjugated linker sequence)CTGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGATGTATACCATCTGGATGCCCGAGAACCCCAGGCCCGGTACCCCCTGCGACATCTTCACCAACAGCAGGGGCAAGCGAGCCAGCAACGGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCTCGGGCATCGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCCGATCCACAACCTCTCGGAGAACCTCCAGCAGCCCCCTCAGGTCTGGGACCTAATACAATGGCTTCAGGCGGTGGCGCTCCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGCACCTCGGGAGGAAGCACCAACGACAACACCTATTTTGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGTCACTTTTCACCACGTGACTGGCAACGACTCATCAACAACAATTGGGGATTCCGGCCCAAAAGACTCAACTTCAAGCTGTTCAACATCCAGGTCAAGGAAGTCACGACGAACGAAGGCACCAAGACCATCGCCAATAATCTCACCAGCACCGTGCAGGTCTTTACGGACTCGGAGTACCAGTTACCGTACGTGCTAGGATCCGCTCACCAGGGATGTCTGCCTCCGTTCCCGGCGGACGTCTTCATGGTTCCTCAGTACGGCTATTTAACTTTAAACAATGGAAGCCAAGCCCTGGGACGTTCCTCCTTCTACTGTCTGGAGTATTTCCCATCGCAGATGCTGAGAACCGGCAACAACTTTCAGTTCAGCTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCCTGGACAGGCTGATGAATCCCCTCATCGACCAGTACCTGTACTACCTGGTCAGAACGCAAACGACTGGAACTGGAGGGACGCAGACTCTGGCATTCAGCCAAGCGGGTCCTAGCTCAATGGCCAACCAGGCTAGAAATTGGGTGCCCGGACCTTGCTACCGGCAGCAGCGCGTCTCCACGACAACCAACCAGAACAACAACAGCAACTTTGCCTGGACGGGAGCTGCCAAGTTTAAGCTGAACGGCCGAGACTCTCTAATGAATCCGGGCGTGGCAATGGCTTCCCACAAGGATGACGACGACCGCTTCTTCCCTTCGAGCGGGGTCCTGATTTTTGGCAAGCAAGGAGCCGGGAACGATGGAGTGGATTACAGCCAAGTGCTGATTACAGATGAGGAAGAAATCAAGGCTACCAACCCCGTGGCCACAGAAGAATATGGAGCAGTGGCCATCAACAACCAGGCCGCCAATACGCAGGCGCAGACCGGACTCGTGCACAACCAGGGGGTGATTCCCGGCATGGTGTGGCAGAATAGAGACGTGTACCTGCAGGGTCCCATCTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCGTCTCCCCTGATGGGCGGCTTTGGACTGAAGCACCCGCCTCCTCAAATTCTCATCAAGAACACACCGGTTCCAGCGGACCCGCCGCTTACCTTCAACCAGGCCAAGCTGAACTCTTTCATCACGCAGTACAGCACCGGACAGGTCAGCGTGGAAATCGAGTGGGAGCTGCAGAAAGAAAACAGCAAACGCTGGAATCCAGAGATTCAATACACTTCCAACTACTACAAATCTACAAATGTGGACTTTGCTGTCAACACGGAGGGGGTTTATAGCGAGCCTCGCCCCATTGGCACCCGTTACCTCACCCGCAACCTGTAASEQ ID NO: 13 AAVrh8VP2Angiopep-2 VP2 nucleotide sequence (includinginserted Angiopep-2):(Underlined sequence indicates peptide and conjugated linker sequence)CTGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGATGACCTTCTTCTACGGCGGCAGCAGGGGCAAGAGGAACAACTTCAAGACCGAGGAGTACGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCTCGGGCATCGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCCGATCCACAACCTCTCGGAGAACCTCCAGCAGCCCCCTCAGGTCTGGGACCTAATACAATGGCTTCAGGCGGTGGCGCTCCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGCACCTCGGGAGGAAGCACCAACGACAACACCTATTTTGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGTCACTTTTCACCACGTGACTGGCAACGACTCATCAACAACAATTGGGGATTCCGGCCCAAAAGACTCAACTTCAAGCTGTTCAACATCCAGGTCAAGGAAGTCACGACGAACGAAGGCACCAAGACCATCGCCAATAATCTCACCAGCACCGTGCAGGTCTTTACGGACTCGGAGTACCAGTTACCGTACGTGCTAGGATCCGCTCACCAGGGATGTCTGCCTCCGTTCCCGGCGGACGTCTTCATGGTTCCTCAGTACGGCTATTTAACTTTAAACAATGGAAGCCAAGCCCTGGGACGTTCCTCCTTCTACTGTCTGGAGTATTTCCCATCGCAGATGCTGAGAACCGGCAACAACTTTCAGTTCAGCTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCCTGGACAGGCTGATGAATCCCCTCATCGACCAGTACCTGTACTACCTGGTCAGAACGCAAACGACTGGAACTGGAGGGACGCAGACTCTGGCATTCAGCCAAGCGGGTCCTAGCTCAATGGCCAACCAGGCTAGAAATTGGGTGCCCGGACCTTGCTACCGGCAGCAGCGCGTCTCCACGACAACCAACCAGAACAACAACAGCAACTTTGCCTGGACGGGAGCTGCCAAGTTTAAGCTGAACGGCCGAGACTCTCTAATGAATCCGGGCGTGGCAATGGCTTCCCACAAGGATGACGACGACCGCTTCTTCCCTTCGAGCGGGGTCCTGATTTTTGGCAAGCAAGGAGCCGGGAACGATGGAGTGGATTACAGCCAAGTGCTGATTACAGATGAGGAAGAAATCAAGGCTACCAACCCCGTGGCCACAGAAGAATATGGAGCAGTGGCCATCAACAACCAGGCCGCCAATACGCAGGCGCAGACCGGACTCGTGCACAACCAGGGGGTGATTCCCGGCATGGTGTGGCAGAATAGAGACGTGTACCTGCAGGGTCCCATCTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCGTCTCCCCTGATGGGCGGCTTTGGACTGAAGCACCCGCCTCCTCAAATTCTCATCAAGAACACACCGGTTCCAGCGGACCCGCCGCTTACCTTCAACCAGGCCAAGCTGAACTCTTTCATCACGCAGTACAGCACCGGACAGGTCAGCGTGGAAATCGAGTGGGAGCTGCAGAAAGAAAACAGCAAACGCTGGAATCCAGAGATTCAATACACTTCCAACTACTACAAATCTACAAATGTGGACTTTGCTGTCAACACGGAGGGGGTTTATAGCGAGCCTCGCCCCATTGGCACCCGTTACCTC ACCCGCAACCTGTAASEQ ID NO: 14 AAV9.47VP1,3 nucleotide sequence (supplies VP1 and VP3in trans to peptide-inserted VP2):ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGGCGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAACTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGATTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACGAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCATAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAASEQ ID NO: 15 AAV9.47VP2ICAMg3 nucleotide sequence (supplies ICAMg3inserted VP2):(Underlined sequence indicates peptide and conjugated linker sequence)CTGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGATGAACAACCAGAAGATCGTGAACCTGAAGGAGAAGGTGGCCCAGCTGGAGGCCGGCGGCGGCGGCAGCGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAACTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGATTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACGAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCATAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAASEQ ID NO: 16 AAV9.47VP2RVG nucleotide sequence (supplies RVG insertedVP2):(Underlined sequence indicates peptide and conjugated linker sequence)CTGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGATGTATACCATCTGGATGCCCGAGAACCCCAGGCCCGGTACCCCCTGCGACATCTTCACCAACAGCAGGGGCAAGCGAGCCAGCAACGGCGGCGGCGGCGGCAGCGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAACTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGATTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACGAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCATAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAASEQ ID NO: 17 AAV9.47VP2Angiopep-2 nucleotide sequence (suppliesAngiopep-2 inserted VP2):(Underlined sequence indicates peptide and conjugated linker sequence)CTGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGATGACCTTCTTCTACGGCGGCAGCAGGGGCAAGAGGAACAACTTCAAGACCGAGGAGTACGGCGGCGGCGGCAGCGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAACTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGATTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACGAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCATAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAASEQ ID NO: 18 AAV9.47VP2A-string nucleotide sequence (supplies A-string inserted VP2):(Underlined sequence indicates peptide and conjugated linker sequence)CTGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGATGGCAGCTGCGGCCGCCGCGGCTGCAGCGGCTGCAGCCGCCGCAGCTGCGGCTGCAGCGGGCGGCGGCGGCAGCGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAACTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGATTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACGAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCATAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAASEQ ID NO: 19 AAVrh8VP2FC5 VP2 amino acid sequence (including insertedFC5):(Underlined sequence indicates peptide and conjugated linker sequence)MEVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYCAAGSTSTATPLRVDYWGKGTQVTVSSGGGGSAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGLGPNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQALGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLVRTQTTGTGGTQTLAFSQAGPSSMANQARNWVPGPCYRQQRVSTTTNQNNNSNFAWTGAAKFKLNGRDSLMNPGVAMASHKDDDDRFFPSSGVLIFGKQGAGNDGVDYSQVLITDEEEIKATNPVATEEYGAVAINNQAANTQAQTGLVHNQGVIPGMVWQNRDVYLQGPIVVAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPLTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL*SEQ ID NO: 20 AAVrh8VP2FC44 VP2 amino acid sequence (includinginserted FC44):(Underlined sequence indicates peptide and conjugated linker sequence)MEVQLQASGGGLVQAGGSLRLSCSASVRTFSIYAMGWFRQAPGKEREFVAGINRSGDVTKYADFVKGRFSISRDNAKNMVYLQMNSLKPEDTALYYCAATWAYDTVGALTSGYNFWGQGTQVTVSSGGGGSAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGLGPNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQALGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLVRTQTTGTGGTQTLAFSQAGPSSMANQARNWVPGPCYRQQRVSTTTNQNNNSNFAWTGAAKFKLNGRDSLMNPGVAMASHKDDDDRFFPSSGVLIFGKQGAGNDGVDYSQVLITDEEEIKATNPVATEEYGAVAINNQAANTQAQTGLVHNQGVIPGMVWQNRDVYLQGPIVVAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPLTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL*SEQ ID NO: 21 AAVrh8VP2ApoB100 VP2 amino acid sequence (includinginserted ApoB100):(Underlined sequence indicates peptide and conjugated linker sequence)MPSSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSPSGGGGSGGGGSAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGLGPNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQALGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLVRTQTTGTGGTQTLAFSQAGPSSMANQARNWVPGPCYRQQRVSTTTNQNNNSNFAWTGAAKFKLNGRDSLMNPGVAMASHKDDDDRFFPSSGVLIFGKQGAGNDGVDYSQVLITDEEEIKATNPVATEEYGAVAINNQAANTQAQTGLVHNQGVIPGMVWQNRDVYLQGPIVVAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPLTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL*SEQ ID NO: 22 AAVrh8VP2RVG VP2 amino acid sequence (including insertedRVG):(Underlined sequence indicates peptide and conjugated linker sequence)MYTIWMPENPRPGTPCDIFTNSRGKRASNGGGGGSGGGGSAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGLGPNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQALGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLVRTQTTGTGGTQTLAFSQAGPSSMANQARNWVPGPCYRQQRVSTTTNQNNNSNFAWTGAAKFKLNGRDSLMNPGVAMASHKDDDDRFFPSSGVLIFGKQGAGNDGVDYSQVLITDEEEIKATNPVATEEYGAVAINNQAANTQAQTGLVHNQGVIPGMVWQNRDVYLQGPIVVAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPLTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL*SEQ ID NO: 23 AAVrh8VP2Angiopep-2 VP2 amino acid sequence (includinginserted Angiopep-2):(Underlined sequence indicates peptide and conjugated linker sequence)MTFFYGGSRGKRNNFKTEEYGGGGSGGGGSAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGLGPNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQALGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLVRTQTTGTGGTQTLAFSQAGPSSMANQARNWVPGPCYRQQRVSTTTNQNNNSNFAWTGAAKFKLNGRDSLMNPGVAMASHKDDDDRFFPSSGVLIFGKQGAGNDGVDYSQVLITDEEEIKATNPVATEEYGAVAINNQAANTQAQTGLVHNQGVIPGMVWQNRDVYLQGPIVVAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPLTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTR YLTRNL*SEQ ID NO: 24 AAV9.47VP2ICAMg3 VP2 amino acid sequence (includinginserted ICAMg3):(Underlined sequence indicates peptide and conjugated linker sequence)MNNQKIVNLKEKVAQLEAGGGGSAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFNYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINDSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADEVMITNEEEIKTTNPVATESYGQVAINHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIVVAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL*SEQ ID NO: 25 AAV9.47VP2RVG VP2 amino acid sequence (includinginserted RVG):(Underlined sequence indicates peptide and conjugated linker sequence)MYTIWMPENPRPGTPCDIFTNSRGKRASNGGGGGSAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFNYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINDSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADEVMITNEEEIKTTNPVATESYGQVAINHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIVVAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEP RPIGTRYLTRNL*SEQ ID NO: 26 AAV9.47VP2Angiopep-2 VP2 amino acid sequence (includinginserted Angiopep-2):(Underlined sequence indicates peptide and conjugated linker sequence)MTFFYGGSRGKRNNFKTEEYGGGGSAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFNYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINDSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADEVMITNEEEIKTTNPVATESYGQVAINHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIVVAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRN L*SEQ ID NO: 27 AAV9.47VP2A-string VP2 amino acid sequence (includinginserted A-string):(Underlined sequence indicates peptide and conjugated linker sequence)MAAAAAAAAAAAAAAAAAAAGGGGSAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFNYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINDSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADEVMITNEEEIKTTNPVATESYGQVAINHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTR NL*SEQ ID NO: 28 Linker sequence: GGGGSSEQ ID NO: 29 Angiopep-2 nucleic acid sequence:ACCTTCTTCTACGGCGGCAGCAGGGGCAAGAGGAACAACTTCAAGACCGAGGAG TAC

This disclosure is not limited in its application to the details ofconstruction and the arrangement of components set forth in thisdescription or illustrated in the drawings. The disclosure is capable ofother embodiments and of being practiced or of being carried out invarious ways. Also, the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting. Theuse of “including,” “comprising,” or “having,” “containing,”“involving,” and variations thereof herein, is meant to encompass theitems listed thereafter and equivalents thereof as well as additionalitems.

Having thus described several aspects of at least one embodiment of thisdisclosure, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe disclosure. Accordingly, the foregoing description and drawings areby way of example only.

1-81. (canceled)
 82. A recombinant AAV (rAAV) comprising a capsidprotein having an N-terminally grafted heterologous targeting peptide,wherein: (i) the capsid protein is a VP2 capsid protein that is not ofan AAV2 serotype; and (ii) the targeting peptide is a CNS-targetingpolypeptide consisting of the amino acid sequence set forth in SEQ IDNO: 5, that is inserted between the first and second amino acid residuesof the VP2 capsid protein.
 83. The rAAV of claim 82, further comprisinga linker conjugated to the C-terminus of the N-terminally graftedheterologous targeting peptide.
 84. The rAAV of claim 83, wherein thelinker comprises at least one polypeptide repeat, each repeat comprisingat least two glycine residues.
 85. The rAAV of claim 84, wherein thelinker is of the formula [G]_(n)S, wherein n is an integer in a range of2 to
 10. 86. The rAAV of claim 84, wherein the linker comprises theformula [GGGGS]_(n), wherein n is an integer in a range of 1 to
 4. 87.The rAAV of claim 86, wherein the linker comprises SEQ ID NO:
 28. 88. Arecombinant AAV (rAAV) comprising a capsid protein having anN-terminally grafted heterologous targeting peptide, wherein: (i) thecapsid protein is a VP2 capsid protein that is not of an AAV2 serotype;and (ii) the targeting peptide is a CNS-targeting polypeptide consistingof the amino acid sequence set forth in SEQ ID NO: 7, that is insertedbetween the first and second amino acid residues of the VP2 capsidprotein.
 89. The rAAV of claim 88, further comprising a linkerconjugated to the C-terminus of the N-terminally grafted heterologoustargeting peptide.
 90. The rAAV of claim 89, wherein the linkercomprises at least one polypeptide repeat, each repeat comprising atleast two glycine residues.
 91. The rAAV of claim 90, wherein the linkeris of the formula [G]_(n)S, wherein n is an integer in a range of 2 to10.
 92. The rAAV of claim 90, wherein the linker comprises the formula[GGGGS]_(n), wherein n is an integer in a range of 1 to
 4. 93. The rAAVof claim 92, wherein the linker comprises SEQ ID NO:
 28. 94. Arecombinant AAV (rAAV) comprising a capsid protein having anN-terminally grafted heterologous targeting peptide, wherein: (i) thecapsid protein is a VP2 capsid protein that is not of an AAV2 serotype;and (ii) the targeting peptide is a CNS-targeting polypeptide encoded bya nucleic acid consisting of the nucleotide sequence set forth in SEQ IDNO: 29, that is inserted between the first and second amino acidresidues of the VP2 capsid protein.
 95. The rAAV of claim 94, furthercomprising a linker conjugated to the C-terminus of the N-terminallygrafted heterologous targeting peptide.
 96. The rAAV of claim 95,wherein the linker comprises at least one polypeptide repeat, eachrepeat comprising at least two glycine residues.
 97. The rAAV of claim96, wherein the linker is of the formula [G]_(n)S, wherein n is aninteger in a range of 2 to
 10. 98. The rAAV of claim 96, wherein thelinker comprises the formula [GGGGS]_(n), wherein n is an integer in arange of 1 to 4, and wherein the linker optionally comprises SEQ ID NO:28.
 99. A method for delivering a transgene to a subject comprising:administering a rAAV of claim 82 to a subject, wherein the rAAVcomprises at least one transgene, and wherein the rAAV infects cells ofa target tissue of the subject.
 100. A method for delivering a transgeneto a subject comprising: administering a rAAV of claim 88 to a subject,wherein the rAAV comprises at least one transgene, and wherein the rAAVinfects cells of a target tissue of the subject.
 101. A method fordelivering a transgene to a subject comprising: administering a rAAV ofclaim 94 to a subject, wherein the rAAV comprises at least onetransgene, and wherein the rAAV infects cells of a target tissue of thesubject.